2,2-Dimethyl-3R-carboxy-6S-acylamido-1-oxa-4-aza-5R-bicyclo[3,2,0]heptan-7-ones

ABSTRACT

The title compounds, which can casually be called 1-oxapenicillins, are produced by total synthesis. Thus 1-oxabenzylpenicillin is produced either by phenylacetylation of 2,2-dimethyl-3R-carboxy-6S-amino-1-oxa-4-aza-5R-bicyclo[3,2,0]heptan-7-one or by hydrolysis and rearrangement of 2-benzyl-6-(1&#39;-methoxycarbonyl-2&#39;-methylprop-1&#39;-enyl)-1-oxa-3,6-diaza-4S, 5R-bicyclo[3,2,0]hept-2-en-7-one.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of my prior, copending application filedApr. 10, 1972 as Ser. No. 242,842 and now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention.

The processes of the present invention produce compounds both old andnew which are useful intermediates in the synthesis of β-lactamantibiotics.

2. Description of the Prior Art.

Penicillins and cephalosporins comprise a group of well-knownantibacterial agents commonly grouped together as a class calledβ-lactam antibiotics. For a recent review of this field with manycitations (especially the first ten) to the prior work see J. P. Hou andJ. W. Poole, β-lactam Antibiotics: Their Physicochemical Properties andBiological Activities in Relation to Structure, J. PharmaceuticalSciences, 60(4), 503-532 (April, 1971). Most of the work in this fieldhas fundamentally been done, speaking broadly, with 6-aminopenicillanicacid, 7-aminocephalosporanic acid and derivatives thereof produced byfermentation.

Considerable work has also been done on total chemical synthesis. Arecent review is the test by M. S. Manhas and A. K. Bose, Synthesis ofPenicillin, Cephalosporin C and Analogues, Marcel Decker, Inc., 95Madison Avenue, New York, New York, 1969. An even more recent review isby R. B. Morin and B. G. Jackson, Chemistry of CephalosporinAntibiotics, Fortschr Chem. Org. Naturst, 28, 343-403 (1970), especiallypages 379-393; the now famous "Woodward Intermediate" is shown thereinas Compound 146 on page 387.

Within recent months publications describing new work and summarizingand citing older work have appeared such as:

A. from Imperial College, London, and Glaxo jointly by D. H. R. Bartonet al., J. Chem. Soc. (C), 1971, 3540-3550;

b. from Oxford University by D. M. Brunwin et al., J. Chem. Soc. (C),1971, 3756-3762;

c. from the University, Newcastle upon Tyne, by B. G. Ramsay and R. J.Stoodley, J. Chem. Soc. (C), 1971, 3859-3867;

d. from Lilly by S. Kukolja, J. Amer. Chem. Soc. 93, 6267-6270 (1971);

E. from Lilly by G. E. Gutowski et al., Tetrahedron Letters No. 37,3433-3436 (1971);

F. from Lilly a series of papers entitled Chemistry of CephalosporinAntibiotics, e.g. No. 25 in J. Medicinal Chemistry 14(11), 1136-1138(1971) and No. 21 in J. Org. Chem., 36(9), 1259-1267 (1971).

The anhydropenicillins used as starting materials in the presentinvention were first described by Saul Wolfe in J. Amer. Chem. Soc. 85,643-644 (March, 1963), Belgium Pat. No. 621,452 and U.S. Patent No.3,311,638. Additional publications on anhydropenicillins by Saul Wolfeinclude Can. J. Chem. 46, 459 and 2549 (1968).

Various azetidinones have been disclosed in patents such as Sheehan'sU.S. Pat. No. 3,487,070-072 (Class 260-239) and 3,487,090 and, forexample, Woodward's U.S. Pat. No. 3,483,215 and U.S. Pat. No. 3,449,336.

The oxazoline having the structure ##STR1## which is more commonlywritten as ##STR2## has been prepared from the methyl ester ofbenzylpenicillin in 18% yield, as by Barton et al., J. Amer. Chem. Soc.,91, 1529 (1969), and (a) above. Only one other bicyclic, sulfur-freecompound of this type has been described [By E. G. Brain et al., J.Chem. Soc. Chem. Comm., 229-230 (1972)] but a rational synthesis of suchesters or acids has not appeared.

The stereochemistry of the methyl ester of benzylpenicillin (i.e., thepenicillin G produced by fermentation) is represented as follows:##STR3##

Kukolja [J. Amer. Chem. Soc. 93, 6267-6270 (1971)] chlorinated (with 2equivalents of chlorine) methyl 6-phthalimidopenicillanate ##STR4## toproduce a mixture of the two epimers having the structure ##STR5##

SUMMARY OF THE INVENTION

This invention provides a series of processes (and certain novelintermediates produced thereby) which begin with the chlorination of ananhydropenicillin.

The processes are set forth schematically in the usual manner directlybelow using for illustrative purposes as the starting material thephthalimido-anhydro-penicillin having the structure ##STR6## Forsimplicity that compound is written below as ##STR7## TFA representstrifluoroacetic acid; DIC is diisopropylcarbodiimide; NBS isN-bromosuccinimide; t-BuOH is tert.-butyl alcohol; Ph is phenyl.

This invention includes, but is not limited to, the reaction schemes setforth below as they are presented solely for purposes of illustration.##STR8##

The above and additional processes and intermediates of the presentinvention are set forth below using as the starting material thetoluenesulfonic acid (TsOH) salt of 6-aminoanhydropenicillin. Thiscompound has the structure. ##STR9## which for convenience isrepresented below as ##STR10##

Thus, direct chlorination (excess Cl₂, CH₂ Cl₂, 20°, 3 min.) ofanhydro-6-phthalimidopenicillin (3a), followed by removal of the solventunder reduced pressure, afforded a 3:2 mixture of two isomericcompounds. The major isomer 4a, m.p 210° dec., was obtained in 38% yield(from 3a) by crystallization from CHCl₃ --petroleum ether (calcd. forC₁₆ H₁₂ N₂ O₄ Cl: C, 52.3; H, 3.31; N, 7.69; Cl, 19.4. Found: C, 52.03;H, 2.96; N, 7.76; Cl, 19.53), λ_(max) ^(FtOH) 242 (10000). The irspectrum of 4a (and of the mixture of 4a and 4b) shows peaks at 5.48(COCl), 5.55 (β-lactam), 5.62, 5.79 β(phthalimido).

Hydrolysis of 4a (boric acid-borax buffer [Saul Wolfe, R. N. Bassett S.M. Caldwell, and F. I. Wasson, J. Amer. Chem. Soc., 91, 7205 (1969)] oraqueous acetone) gave the acid 4c, m.p. 168°-174° dec. in 73% yield(calcd. for C₁₆ H₁₃ N₂ O₅ Cl: C, 55.05; H, 3.76; N, 8.02; Cl, 10.02;Found: C, 54.65; H, 3.79; N, 8.46; Cl, 10.53); λ_(max) ^(FtOH) 236(7400), 296 (1800); λ_(max) ^(KBr) 5.52, 5.60, 5.80, 5.85μ. Methylationof 4c (CH₂ N₂, Et₂ O--CH₂ Cl₂) produced the ester 4e, m.p. 178°-180°dec. in 97% yield. The same compound was obtained from 4a in 93% yieldupon treatment with absolute methanol (30 min., 20°). Treatment of themixture of 4a and 4b with methanol (30 min., 20°) and crystallization ofthe resultant residue (CHCl₃ -petroleum ether) afforded 4e in 55% yield.Chromatography of the residual mother liquor (Woelm II alumina; 1:1 CH₂Cl₂ :C₆ H₆) produced 22% of 4f, the methyl ester derivative of 4b, m.p.192°-194°, in the first fractions followed by an additional 9% of 4e.The t-butyl ester 4g was obtained in 33% yield by refluxing 4a inanhydrous t-BuOH for 21 hr. Dissolution of 4g in trifluoroacetic acidand evaporation of the solvent after 3 min. gave 4a in quantitativeyield.

The relative amounts of 4a and 4b were not significantly affected bychlorination in the presence of excess Et₄ N^(+Cl) ^(-;) otherchlorinating agents (SO₂ Cl₂, pyridinium trichloride, pyrrolidonehydrotrichloride) gave more complex mixtures. The esters 4e and 4f wereequilibrated [Under identical experimental conditions the same mixturewas obtained from both directions] upon refluxing (Me₂ CO, 12 hr.;2-butanone, 4 hr.) with excess Et₄ N^(+Cl) ⁻, the trans isomer 4fpredominating. Depending on the conditions, ratios varying from 3:2 to5:2 could be obtained and the isomers could be separated bychromatography, as described above.

Reaction of 4e with sodium azide [4c decomposed when subjected to theseconditions] (DMF, 90°, 3 hr.) afforded the trans azide 4h, m.p.144°-145° (67%); under the same conditions 4f was converted to 4i, m.p.183°-187° (52%). Reaction of 4f with Et₄ N^(+OAc) ⁻ (pure CHCl₃, reflux,17 hr.) gave the cis-acetate 4j (62%). Reaction of 4f withtetramethylguanidinium formate [a reagent invented for this purpose]afforded the cis-formate 4k (35%)

The above sequence of reactions was then repeated, with variousmodifications, on the p-toluenesulfonic acid salt [S. Wolfe, Can. J.Chem., 46, 459 (1968)] of anhydro-6-aminopenicillin (3b). Chlorination(0°-5°, CH₂ Cl₂), followed by removal of the solvent and triturationwith ether, afforded a 4:1 mixture of 5a and 5b as a stable (below 20°)white powder, m.p. 148°-149° dec. Hydrolysis of this powder(acetone-water) gave a 4:1 mixture of the acids 5c and 5d.Alternatively, methanol treatment of the mixture of 5a and 5b, andcrystallization from chloroform-hexane produced the pure cis-methylester 5e, m.p. 125°-130° dec., in 67% yield. Neutralization of 5e(NaHCO₃) and reaction of the free base 6a with N-carbethoxyphthalimide[G. H. L. Nefkens, Nature, 185, 309 (1960)] gave 4e to provideconfirmation that the chlorination reaction had proceeded in the samemanner with the two anhydropenicillins.

Equilibration of 6a with the trans isomer 6b proceeded smoothly in thepresence of chloride ions, the optimum ratio in favor of 6b (6:1) beingachieved with tetramethylguanidinium chloride (CH₂ CL₂, 12 hr. reflux).The isomers were separated by alumina chromatography. The cis aminochloride 6a reacted smoothly with tetramethylguanidinium azide [A. J.Papa, J. Org. Chem., 31, 1426 (1966)] (2 equiv., CHCl₃, 1 hr. reflux) togive the trans amino azide 6d (90%); the cis amino azide 6c, m.p.116°-117°, was obtained similarly from 6b in 85% yield.

The 5-chloropenicillin G analog 7a, m.p. 111°-115° dec., was obtained(82%) upon phenylacetylation of 6a (PhCH₂ COOH, diisopropylcarbodiimide(DIC), CH₂ Cl₂). The C5 epimer 7b, obtained similarly from 6b, cyclizedspontaneously to the oxazoline 2 [J. C. Sheehan, "Molecular Modificationin Drug Design", Advances in Chemistry Series, No. 45, American ChemicalSociety, Washington, D.C., 1964, page 15.; D. H. R. Barton, F. Comer andP. G. Sammes, J. Amer. Chem. Soc., 91, 1529 (1969)], m.p. 126.5-127°;additional quantities of 2 were obtained from 7a by rapid chromatographyon silica gel or alumina [If the trans-acylamino chloride 7b was shakenwith bicarbonate varying quantities (up to 10%) of 7e could be isolatedin addition to the oxazoline]. The 5-azidopenicillin G analog 7c, m.p.102°-103°, was obtained (83 %) upon phenylacetylation of 6c (PhCH₂ COOH,DIC, CH₂ Cl₂); the C5 epimer 7d was obtained similarly from 6d or byreaction of 7a with tetramethylguanidinium azide (CHCl₃, reflux).

Allylic oxidation of 4e by N-bromosuccinimide (NBS, 2 equiv., CCl₄,benzoyl peroxide, Photoflood No. 2 bulb [S. Wolfe, and D. V. C. Awang,Can. J. Chem., 49, 1384 (1971); This result should be contrasted to thebehavior of the parent anhydropenicillin towards allylic oxidation; S.Wolfe, C. Ferrari and W. S. Lee, Tetrahedron Letters, 3385 (1969). Asalready noted, S. Wolfe, R. N. Bassett, S. M. Caldwell, and F. I.Wasson, J. Amer. Chem. Soc., 91, 7205 (1969), enamine character of thedouble bond of these compounds is not seen in the monocyclic systems])was complete within 5 min. The product 8a crystallized from CCl₄-petroleum ether in 81% yield, m.p. 70°-72° dec. Under the sameconditions the C5 epimer 4f afforded 8b (70%). The extraordinaryfacility of this NBS oxidation is seen in the successfulfunctionalization, under the same conditions, of 7c and 2. The methylgroups of 4e and 4f show no significant difference in reactivity. Thus,oxidation of 4e with one equivalent of NBS afforded a 1:1 mixture of 8cand 8d. Treatment of this mixture with tetramethylguanidinium azide (oneequivalent, CHCl₃, 3 hr., 20° [The 5-chloro substituent is stable underthese conditions.]) afforded a 1:1 mixture of 8e and 8f; λ_(max) ^(CHCl)3 4.7 (azide), 5.55 (β-lactam), 5.62, 5.79 (phthalimido), 5.78 (ester).Hydrogenation (PtO₂, benzene, 45 psi, 6 hr.) yield the mixture of amines8g and 8h; λ_(max) ^(CHCl) 3 5.55, 5.62, 5.79, 5.83 μ. This mixture wasrecovered unchanged following refluxing in chloroform. Treatment withKOtBu-tBuOH (20°, 1 hr.), followed by carful chromatography (neutralalumina, activity II, elution with graded mixtures of C₆ H₆ --CH₂ Cl₂)produced in 12% yield (24% if only one of 8g or 8h reacted) acrystalline compound, m.p. 121°-122° having structure 9 on the basis ofits ir (λ_(max) ^(KBr) 2.92, 5.56, 5.62, 5.79, 5.82, 6.03 μ) and nmr(2.25 (4H), 3.87 (1H,d,2.0 Hz), 4.30 (1H,d,2.0 Hz), 6.23 (3H), 7.70(2H), 7.88 (3H)) spectra.

Another important embodiment of the present invention is schematicallyillustrated, with R indicating n-propyl or tert. butyl and ##STR11##

More generally speaking, the processes described above and in theexamples are conducted with numerous side-chains at the 6-position, e.g.using as the original reagent as anhydropenicillin having the formula##STR12## wherein R¹ represents acylamino, including phthalimido, oramino.

Thus in the processes herein disclosed use is also made in place of thephthalimido group (Ft) or the free amino group or its tosylate (Ts) ofthe corresponding reagents, intermediates and products in which each ofthese groups is replaced by an acylamino group R¹ of the formula##STR13## wherein R⁴ represents a member selected from the groupconsisting of hydrogen, amino, carbobenzoxyamino, phenyl, fluoro,chloro, bromo, iodo, hydroxy, (lower)alkanoyloxy and (lower)alkoxy; Xrepresents a member selected from the group consisting of oxygen andsulfur; R⁵ and R⁶ each represent a member selected from the groupconsisting of hydrogen, phenyl, benzyl, phenethyl and (lower)aklyl; R⁷represents (lower)alkyl; R⁸ and R⁹ each represent a member selected fromthe group consisting of (lower)alkyl, (lower)alkylthio, benzylthio,cyclohexyl, cyclopentyl, cycloheptyl, benzyl, phenetyl, phenylpropyl,furyl, thienyl, naphthyl and Ar--; R¹⁰ represents a member selected fromthe group consisting of (lower)alkylamino, di(lower)alkylamino,cycloalkylamino having from 3 to 7 carbon atoms inclusive, allylamino,diallylamino, phenyl(lower)alkylamino, morpholino,lower(alkyl)morpholino, di-(lower)alkylmorpholino,morpholino(lower)alkylamino, pyrrolidino, (lower)alkylpyrrolidino,di(lower)alkylpyrrolidino, N,N-hexamethyleneimino, piperidino,(lower)alkylpiperidino, di(lower)-alkylpiperidino,1,2,5,6-tetrahydropyridino, N-(lower)alkylpiperazino,N-phenylpiperazino, N-(lower)alkyl(lower)alkylpiperazino,N-(lower)alkyl-di-(lower)alkylpiperazino, furfurylamino,tetrahydrofurfurylamino, N-(lower)alkyl-N-furfurylamino,N-alkyl-N-aniline and (lower)alkoxyanilino; Z¹, Z² and Z³ each representa member selected from the group consisting of (lower)alkyl and Ar--;R¹¹ represents a member selected from the group consisting of(lower)alkyl, (lower)cycloalkyl, naphthyl, benzyl, phenethyl and##STR14## R¹² represents 2,2,2-trichloroethyl or benzyl andAr--represents a monovalent radical having one of the formula whereinR¹, R² and R³ are each a member selected from the group consisting ofhydrogen, chloro, bromo, iodo, trifluoromethyl, phenyl, (lower)alkyl and(lower)alkoxy, but only one of said R¹, R² and R³ may represent phenyl.

Thus, for example, a preferred group of intermediates and final productsof the present invention are illustrated in terms of final products byacids having the following formulae: ##STR15## wherein R represents(lower)alkyl; ##STR16## wherein R¹ represents (lower)alkyl and R² and R³each represent a member selected from the group consisting of hydrogenand chloro; ##STR17## wherein R¹ is (lower)alkyl and R² is a memberselected from the group consisting of hydrogen and chloro; ##STR18##wherein R represents (lower)alkyl; ##STR19## wherein R is (lower)alkyl;##STR20## wherein R is (lower)alkyl; ##STR21## wherein R represents(lower) alkyl; ##STR22## wherein R¹ represents (lower)alkyl and R² andR³ each represent a member selected from the group consisting ofhydrogen and chloro; ##STR23## wherein R¹ is (lower)alkyl and R² is amember selected from the group consisting of hydrogen and chloro;##STR24## wherein R represents (lower)alkyl; ##STR25## wherein R is(lower)alkyl; ##STR26## wherein R is (lower)alkyl.

The present invention provides the process for the production of anantibacterial agent which comprises reacting a compound of the formula##STR27## or a salt thereof with an organic monocarboxylic acid chlorideor a functional equivalent thereof, and also the process for theproduction of an antibacterial agent which comprises reacting a compoundof the formula ##STR28## or a salt thereof with an organicmonocarboxylic acid chloride or a functional equivalent thereof.

In a preferred embodiment the present invention provides the process forthe production of an antibacterial agent which comprises reacting acompound of the formulae ##STR29## or a salt thereof either with anorganic monocarboxylic acid chloride of the formula ##STR30## wherein R¹represents a member selected from the group consisting of hydrogen,amino, carbobenzoxyamino, phenyl, fluoro, chloro, bromo, iodo, hydroxy,(lower)alkanoyloxy and (lower)alkoxy; X represents a member selectedfrom the group consisting of oxygen and sulfur; R⁵ and R⁶ each representa member selected from the group consisting of hydrogen, phenyl, benzyl,phenethyl and (lower)alkyl; R⁷ represents (lower)alkyl; R⁸ and R⁹ eachrepresent a member selected from the group consisting of (lower)alkyl,(lower)alylthio, benzylthio, cyclohexyl, cyclopentyl, cycloheptyl,benzyl, phenethyl, phenylpropyl, furyl, thienyl, naphthyl and Ar--; R¹⁰represents a member selected from the group consisting of(lower)alkylamino, di(lower)alkylamino, cycloalkylamino having from 3 to7 carbon atoms inclusive, allylamino, diallylamino,phenyl(lower)alkylamino, morpholino, (lower)alkylmorpholino,di(lower)alkylmorpholino, morpholino(lower)alkylamino, pyrrolidino,(lower)alkylpyrrolidino, di(lower)alkylpyrrolidino,N,N-hexamethyleneimino, piperidino, (lower)alkylpiperidino,di(lower)alkylpiperidino, 1,2,5,6-tetrahydropyridino,N-(lower)alkylpiperazino, N-phenylpiperazino,N-(lower)alkyl(lower)alkylpiperazino,N-(lower)-alkyl-di(lower)alkylpiperazino, furfurylamino,tetrahydrofurfurylamino, N-(lower)alkyl-N-furfurylamino,N-alky-N-anilino and (lower)alkoxyanilino; Z¹, Z² and Z³ each representa member selected from the group consisting of (lower)alkyl and Ar--;R¹¹ represent a member selected from the group consisting of(lower)-alkyl, (lower)cycloalkyl, naphthyl, benzyl, phenethyl and##STR31## and Ar- represents a monovalent radical having one of theformulae ##STR32## wherein R¹, R² and R³ are each a member selected fromthe group consisting of hydrogen, chloro, bromo, iodo, trifluoromethyl,phenyl, (lower)alkyl and (lower alkoxy, but only one R group mayrepresent phenyl; or a functional equivalent of said acid chloride.

The term (lower)alkyl as used herein means both straight and branchedchain aliphatic hydrocarbon radicals having from one to ten carbon atomssuch as methyl, ethyl, propyl, isopropol, butyl, isobutyl, t-butyl,amyl, hexyl, 2-ethylhexyl, heptyl, decyl, etc. Similarly, where the term(lower) is used as part of the description of another group, e.g.(lower)alkoxy, it refers to the alkyl portion of such group which istherefore as described above in connection with (lower)alkyl.

The functional equivalents of the above acid chlorides as an acylatingagent for a primary amino group include the corresponding carboxylicacid bromides, acid anhydrides, including mixed anhydrides andparticularly the mixed anhydrides prepared from stronger acids such asthe lower aliphatic monoesters of carbonic acid of alkyl and arylsulfonic acids and of more hindered acids such as diphenylacetic acid.In addition, an acid azide or an active ester or thioester (e.g. withp-nitrophenol, 2,4-dinitrophenol, thiophenol, thioacetic acid) may beused or the free acid itself may be coupled with the primary amine afterfirst reacting said free acid with N,N'-dimethylchloroformimiminiumchloroide [cf. Great Britain 1,008,170 and Novak and Weichet,Experientia XXI/6, 360 (1965)] or by the use of enzymes or of anN,N'-carbonyldiimidazole or an N,N'-carbonylditriazole [cf. SouthAfrican Patent Specification 63/2684], of a carbodiimide reagent[especially N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimideor N-cyclohexyl-N'-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide; cf.Sheehan and Hess, J. Amer. Chem. Soc. 77, 1067, (1955)], or ofalkynylamine reagent [cf. R. Buifle and H.G. Viehe, Angew. Chem.International Edition 3, 582 (1964)], or of a ketenimine reagent [cf. C.L. Stevens and M.E. Monk, J. Amer. Chem. Soc. 80, 4065 (1958)] or of anisoxazolium salt reagent [cf. R. B. Woodward, R. A. Olofson and H.Mayer, J. Amer. Chem. Soc. 83, 1010 (1961)]. Another equivalent of theacid chloride is a corresponding azolide, i.e. an amide of thecorresponding acid whose amide nitrogen is a member of a quasi-aromaticfive-membered ring containing at least two nitrogen atoms, i.e.imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole andtheir substituted derivatives. As an example of the general method forthe preparation of an azolide, N,N'-carbonyldiimidazole diimidazole isreacted with a carboxylic acid in equimolar proportions at roomtemperature in tetrahydrofuran, chloroform, dimethylformamide or asimilar inert solvent to form the carboxylic acid imidazolide inpractically quantitative yield with liberation of carbon dioxide and onemole of imidazole. Dicarboxylic acids yield diimidazolides. Theby-product, imidazole, precipitates and may be separated and theimidazolide isolated but this is not essential. The methods for carryingout these reactions to produce a penicillin and the methods used isisolate the pencillins so-produced are well-known in the art.

The nontoxic, pharmaceutically acceptable salts include metallic saltssuch as sodium, potassium, calcium and aluminum, the ammonium salt andsubstituted ammonium salts, e.g. salts of such nontoxic amines astrialkylamines, including triethylamine, procaine, dibenzylamine,N-benzyl-beta-phenethylamin, 1-ephenamine, N,N'-dibenzylethylenediamine,dehydroabietylamine, N,N'-bis-dehydroabietylethylenediamine,N-(lower)alkylpiperidines, e.g. N-ethylpiperidine, and other amineswhich have been used to form salts with benzylpenicillin.

There are particularly included within the scope of the presentinvention the antibacterial agents which are prepared in theabove-described acylation process by the use therein of the organicmonocarboxylic acids or their acid chlorides or other equivalents whichhave previously been used to acylate 6-amino-penicillanic acid asdescribed, for example, in U.S. Pat. Nos. 2,941,995; 2,951,839;2,985,648; 2,996,501; 3,007,920; 3,025,290; 3,028,379; 3,035,047;3,040,032; 3,040,033; 3,041,332, 3,041,333; 3,043,831; 3,053,831;3,071,575; 3,071,576; 3,079,305; 3,079,306; 3,080,356; 3,082,204;3,093,547; 3,093,633; 3,116,285; 3,117,119; 3,118,877; 3,120,512;3,120,513; 3,120,514; 3,127,394, 3,140,282; 3,142,673; 3,147,247;3,174,964; 3,180,863; 3,198,804; 3,202,653; 3,202,654; 3,202,655;3,210,337; 3,157,639; 3,134,767; 3,132,136; in British PatentSpecifications 874,414; 874,416; 876,516; 876,662, 877,120; 877,323;877,531, 878,233; 880,042; 880,400; 882,335; 888,110; 888,552; 889,066;889,069; 889,070; 889,168; 889,231; 890,210; 891,174; 891,279; 891,586;891,777; 891,938; 893,518; 894,247; 894,457; 894,460; 896,072; 899,199;900,666; 902,703; 903,785; 904,576; 905,778; 906,383; 908,787; 914,419;916,097; 916,204; 916,205; 916,488; 918,169; 920,176; 920,177; 920,300;921,513; 922,278; 924,037; 925,281; 931,567; 932,644; 938,066; 938,321;939,708; 940,488; 943,608; 944,417; in numerous Belgian Patents, e.g.593,222; 595,171; 597,859; 602,494; 603,703; 609,039; 616,419; 617,187;and in South African Patent Applications, e.g. 60/2882; 60/3057;60/3748; 61/1649; R61/2751; 62/54; 62/4920; 63/1612 and 63/2423.

When the acylamino group of the compounds of the present invention alsocontains a strongly basic group, e.g. primary amino, as in the case ofthe preferred embodiments having the formulae ##STR33## wherein Ar is asdefined above (and R¹, R² and R³ in Ar are preferably hydrogen), theproducts are amphoteric and normally exist in the zwiteterrion form butcan form acid addition salts, as with such nontoxic, pharmaceuticallyacceptable organic salts as acetic, citric, succinic, ascorbic and thelike and with inorganic acids such as hydrochloric, hydrobromic,sulfuric, phosphoric and the like.

The processes of the present invention also include the use as "finalreagents" of the compound of the formula ##STR34## and its salts withacids and bases and the compound of the formula ##STR35## and its saltswith acids and bases.

These final reagents are prepared, for example, by subjecting tohydrogenolysis the compound of the formula ##STR36## or by subjecting tohydrogenolysis the compound of the formula ##STR37## or by subjecting toacid cleavage either the compound of the formula the compound of theformula ##STR38## or by treating with zinc and acetic acid the compoundof the formula ##STR39## or the compound of the formula ##STR40##

Such esters are prepared, for example, by using as the starting materialan anhydropenicillin of the formula ##STR41## in which R¹ is ##STR42##formyl, o-nitrophenylsulfenylamino or o-nitrophenoxyacetamido or one ofits equivalents as described in U.S. Pat. No. 3,271,409; and in theappropriate later step (of reaction with acid chloride) substituting forthe methanol either t-butyl alcohol or trityl alcohol or trichloroethylalcohol; or in the later step (of reaction with carboxylic acid)substituting for the diazomethane either diphenyldiazomethane,2,2,2-trichloroethyl chloroformate, benzyl chloroformate, or t-butylchloroformate.

These esters are then converted to final reagents by removal of theester and conversion of the blocked amino group to a free amino group,either consecutively or simultaneously. Thus, when the blocked aminogroup represents phthalimido, it is converted to primary amino bytreatment with hydrazine hydrate in dioxane at about room temperaturefor at least twelve hours at with methylamine in aqueous dioxane. Whenthe blocked amino group represents carbobenzyloxyamino it is convertedto a primary amino group by catalytic hydrogenation to leave theproduct. When the blocked amino group is formyl, carbo-t-butyloxy-aminoor o-nitrophenylsulfenylamino it is converted to primary amino either byreaction with anhydrous hydrogen chloride in a nonprotonating solventsuch as benzene or methylene chloride or by reaction withtrifluoroacetic acid. The trichloroethoxycarbonylamino is converted toprimary amino by reaction with zinc in aqueous acetic acid. In addition,when the blocked amino group is carbo-t-butyloxyamino, the final tworeactions are conducted in one step by the use of stronger acid forlonger periods of time and, if desired, at higher temperatures.

When the blocked amino group is o-nitrophenoxyacetamido, it is convertedinto a primary amino group by either (a) catalytic hydrogenation (e.g.in water at room temperature using 30% Pd-on-diatomaceous earth)followed by allowing the mixture to stand at an acidic pH (e.g. in wateracidified to pH 2 with 20% hydrochloric acid at about 10° C. for atleast 20 minutes or at 25° C. for at least 24 hours) or (b) by addingthe blocked compound (e.g. 7 millimoles) in cold water, e.g. 30 ml.,rapidly, e.g. over 1-3 minutes, to 5% Pd-C (e.g. 0.05 g.) suspended in acold solution of KBH₄, (e.g. 14 millimoles) dissolved in water, e.g. 70ml.

Chlorination of Anhydro-6-Phthalimidopenicillin

Chlorine gas was passed at room temperature into a solution ofanhydro-6-phthalimidopenicillin (500 mg., 1.52 mmoles) in methylenechloride (15 ml.). The gas introduction was terminated after 3 minutesand, after an additional 5 minutes, the yellow solution was evaporatedto dryness. The resulting white foam was crystallized from a mixture ofchloroform and petroleum ether to give a total of 250 mg. (32%) ofmaterial in two crops, m.p. 210° dec. The IR spectrum of th residualmother liquor was identical to that of the crystalline material,summarized below.

Anal. Calcd. for C₁₆ H₁₂ N₂ O₄ Cl₂ : C, 52.3; H, 3.31; N, 7.69; Cl,19.4.

Found: C, 53.03; H, 2.96; N, 7.76; Cl, 19.53.

The IR spectrum shows peaks in the carbonyl region at 5.48, 5.55, 5.62and 5.79μ. The NMR spectrum shows peaks at 2.13 (4H), 3.78(1H,d,J=4.2Hz), 4.24 (1H,d,J=4.2 Hz), 7.60 (3H), 7.69 (3H). The uv spectrum showsμmax FtOH 242 (10,000).

The compound has the structure ##STR43##

HYDROLYSIS OF THE DICHLORIDE

The dichloride (500 mg., 1.36 mmoles), in dimethyl sulfoxide, (500 ml.)was treated, dropwise at 0°, with 50 ml. of a pH 7.95 buffer (preparedby diluting 50 ml. of 0.1M boric acid and 2.6 ml. of 0.1M NaOH to 100ml.). At the end of the addition, the pH of the reaction mixture was6.8. This mixture was allowed to warm to room temperature, and stirringwas continued for 3.25 hours. Then ice-cold water (300 ml.) was added,the mixture brought to pH 3 with ice-cold N HCl, and extracted with five100 ml. portions of methylene chloride. The combined extracts werewashed twice with water (100 ml. portions) and dried over anhydroussodium sulfate. Evaporation under reduced pressure at 40° afforded amobile liquid which contained a considerable amount of DMSO. This wasredissolved in chloroform (60 ml.) and the solution washed ten timeswith water (10 ml. portions). After drying and evaporation there wasobtained from the chloroform phase 0.59 g. of a crystalline white solid.This was recrystallized from a mixture of chloroform and petroleum ethergiving a total of 350 mg. (73%) of short white needles, m.p. 168°-174°dec.

Anal. Calcd. for C₁₆ H₁₃ N₂ O₅ Cl: C, 55.05; H, 3.76; N, 8.02; Cl,10.02. Found: C, 54.65; H, 3.79; N, 8.46; Cl, 10.53.

The IR spectrum shows peaks in the carbonyl region at 5.52, 5.60, 5.80and 5.85μ. The NMR spectrum shows peaks at 2.13 (4H), 3.78 (1H,d,J = 4.1Hz), 4.29 (1H,d,J = 4.1 Hz), 7.67 (3H), 7.69 (3H). The u.v. spectrumshows λ _(max) ^(EtOH) 236 (7400), 296 (1800).

The compound has the structure ##STR44##

Conversion of the Acid to the Methyl Ester

The acid (200 mg., 0.57mmole) was dissolved in 30 ml. of a 1:1 mixtureof methylene chloride and ether. The solution was cooled and treatedwith excess ethereal diazomethane. The solution which resulted wasstirred for 0.5 hour, allowed to warm to room temperature during anadditional 1 hour, and then evaporated at 40° to a white crystallineresidue. Recrystallization from a mixture of chloroform and petroleumether gave 0.20 g. (97%) of colorless fine needles, m.p. 178°-180° dec.

The compound has the structure ##STR45##

Conversion of the Acid Chloride to the Methyl Ester

The crystalline acid chloride (500 mg., 143 mmoles) was dissolved inmethylene chloride (10 ml.) and absolute methanol (10 ml.) was added.The mixture was stirred at room temperature for 30 minutes, then dilutedwith methylene chloride (50 ml.) and washed successively with ice-cold5% bicarbonate solution (10 ml.) and water. After drying over anhydrousmagnesium sulfate and removal of the solvent a crystalline residue wasobtained. This was recrystallized from methylene chloride - petroleumether to give 463 mg. (93%) of methyl ester identical to that describedabove.

Anal. Calcd. for C₁₇ H₁₅ N₂ O₅ Cl: C, 56.3; H, 4.16; N, 7.71; Cl, 9.78.Found: C, 56.58; H, 4.37; N, 7.55; Cl, 9.82.

The NMR spectrum shows peaks at 2.10 (m,4H), 3.77 (1H,d,J=4.1 Hz), 4.23(1H,d,J=4.1 Hz), 6.18 (3H), 7.57 (3H), 7.66 (3H).

Direct Conversion of Anhydro-6-Phthalimidopenicillin Into A Mixture ofMethyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoate andits 2'S epimer

Chlorine gas was passed at room temperature into a solution ofanhydro-6-phthalimidopenicillin (1.0 g., 3.04 mmoles) in methylenechloride (30 ml.). After 3 min. the gas introduction was terminated and,after an additional 5 min. the solvent was removed under reducedpressure at 30°. The solid residue was redissolved in methylene chloride(20 ml.) and absolute methanol (20 ml.) was added. This solution wasstirred at room temperature for 30 min. and then evaporated to drynessunder reduced pressure at 40°. The residue was crystallized fromchloroform-petroleum ether to give 600 mg. (55%) of the ester having the2'R-chloro configuration. The mother liquor was chromatographed on a 1.5× 14 cm column of alumina (Woelm, grade II). Elution with 1:1 methylenechloride - benzene afforded 202 mg. (18%) of a new compound in the first25 ml., followed by 93 mg. of a mixture of the 2'R-ester and the newcompound in the next 25 ml. Elution with methylene chloride (40 ml.)then afforded an additional 68 mg. of the 2'R-ester.

The new compound was crystallized from methylene chloride-petroleumether to give the ester having the 2'S-chloro configuration, m.p.192°-194° .

Anal. Calcd. for C₁₇ H₁₅ N₂ O₅ Cl: C, 56.3; H, 4.16; N, 7.71; Cl, 9.78.Found: C, 56.42; H, 4.43; N, 7.68; Cl, 9.60.

The NMR spectrum shows peaks at 2.17 (4H,m), 3.77 (1H,d,J=2.0 Hz), 4.4(1H,d,J= 2.0 Hz), 6.17 (3H), 7.7 (3H), 7.92 (3H).

The structure of this compound is ##STR46##

Stereoselectivity of the Chlorination of Anhydro-6-Phthalimidopenicillin

Anhydro-6-phthalimidopenicillin (0.5 g., 1.52 mmoles) was chlorinated inmethylene chloride, as already described. The NMR spectrum of the totalproduct showed peaks at 2.12 (4H,m), 3.78 (0.6H,d,J=4.2 Hz), 3.77(0.4H,d,J=1.8 Hz), 4.25 (0.6H,d,J=4.2 Hz), 4.33 (0.4H,d,J=1.8 Hz), 7.57(1.8H), 7.65 (1.2H), 7.68 (1.2H), 7.77 (1.2H).

The chlorination reaction is therefore stereoselective under theseconditions, a 60:40 mixture of the 2'R-chloro: 2'S-chloro epimers beingobtained under these conditions.

Crystallization of the product from methylene chloride-petroleum etherafforded 300 mg. (38%) of the pure dichloride having the2'R-configuration. The mother liquor was then dissolved in absolutemethanol (10 ml.) and the solution allowed to stand for 30 min. at roomtemperature with occasional shaking. It was then diluted with methylenechloride and washed successively with ice-cold sodium bicarbonatesolution and water, and dried over anhydrous magnesium sulfate.Evaporation of the solvent gave a residue whose NMR spectrum showed itto be a 1:2 mixture of 2'R- and 2'S-methyl esters. This waschromatographed on alumina (Woelm; grade II), elution with 1:1benzene-methylene chloride afforded 120 mg. of the 2'S-methyl ester inthe first 10 ml. of eluate, followed by mixture of the 2'R and 2'Sesters. The first eluate was crystallized from methylenechloride-petroleum ether to give 55 mg. (7%) of pure 2'S ester.

In a second experiment the chlorination was performed for 3 min., asalready described and the solution allowed to stand at room temperaturefor 3 hr. Evaporation then afforded a mixture containing 68% of2-(2'S-chloro-3'S -phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoylchloride and 32% of the 2'R epimer, i.e., under these conditions thecis:trans ratio was revised in favor of the trans isomer. The same 68:32ratio was obtained if chlorination was performed for 30 seconds and thesolution immediately evaporated to dryness.

When chlorination was performed for 10 seconds with a slow stream ofchlorine, and the mixture then immediately evaporated to dryness, asingle new compound was obtained. The NMR spectrum of this new compoundshowed peaks at (CDCl.sub. 3) 2.1 (4H,d), 4.01 (2H,s), 7.60 (3H), 7.64(3H). In CD₃ COCD₃ the two-proton singlet at 4.01 showed slightsplitting to a doublet. The compound is assigned the structure ##STR47##That it is an intermediate in the chlorination of the anhydropenicillinwas shown by its conversion, upon further chlorination to the 2'R and2'S dichlorides.

Equilibration of methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoate andits 2'S epimer

A. Starting with the 2'R isomer.

The pure 2'R ester (100 mg., 0.28 mmole) was added to a warm solution oftetraethylammonium chloride (95.2 mg., 0.57 mmole; two molarequivalents) in acetone (15 ml.). The solution was refluxed for 2 hours,and then cooled to room temperature and poured onto a mixture of waterand ice. Extraction with methylene chloride, followed by washing of theextract with water, drying over anhydrous magnesium sulfate andevaporation afforded a crystalline residue. The NMR spectrum of thisresidue was identical to that of the starting material except for theappearance of a weak doublet at τ 4.4 (J=2 Hz). The substance wastherefore redissolved in acetone containing tetraethylammonium chloride(95.2 mg.), and refluxing was continued for an additional 14 hours.Isolation in the same manner as described above afforded a residue whoseNMR spectrum showed it to be a 1:1 mixture of 2'R and 2'S esters.

The experiment was repeated with 100 mg. (.28 mmole) of the pure 2'Rester and 238 mg. (5 molar equivalents) of tetraethylammonium chloridein methyl ethyl ketone (10 ml.). After 4 hours of refluxing the productwas isolated and found to be a 3:2 mixture of 2'R and 2'S esters. Thisratio did not change when the material was refluxed for a further 8.5hours in methyl ethyl ketone containing 5 molar equivalents oftetraethylammonium chloride.

The experiment was repeated with 100 mg. of the pure 2'R ester inacetone (15 ml.) containing 5 molar equivalents of tetraethylammoniumchloride. After 12 hours and after 24 hours refluxing a 3:2 2'S:2'Rratio of esters was observed. The product of a 12 hour reaction waschromatographed on alumina (Woelm, grade II). Elution withbenzene-methylene chloride (1:1), as already described, afforded 40 mg.of the 2'S compound in the first 25 ml. followed by 34 mg. of the 2'Scompound in the next 45 ml. of eluate.

After 6 hours in refluxing chloroform (30 ml.) containing 5 molarequivalents of tetraethylammonium chloride, the 2'R ester (430 mg.) wasconverted into a 2:1 2'R:2'S mixture.

B. Starting with the 2'S isomer

The 2'S ester (40 mg.) was refluxed for 12 hours in a mixture of acetone(10 ml.) and methylene chloride (5 ml.) containing 2 molar equivalentsof tetraethylammonium chloride. The product was a 1:2 2'R:2'S mixture.

Chlorination of Anhydro-6-Phthalimidopenicillin in the Presence ofTetraethylammonium Chloride

A solution of anhydro-6-phthalimidopenicillin (100 mg., 0.30 mmole) inmethylene chloride (5 ml.) containing tetraethylammonium chloride (252mg. 5 molar equivalents) was chlorinated in the usual manner.Evaporation of the solvent then gave a residue which consisted of a 3:2mixture of 2'S: 2'R dichlorides.

The experiment was repeated using 10 ml. of methylene chloride for thereaction. After removal of the solvent, absolute methanol (5 ml.) andmethylene chloride (5 ml.) were added and the mixture was stirred for 30min. Then it was washed with ice-cold sodium bicarbonate solution andwater, dried over magnesium sulfate, and evaporated. The residue (132mg.) was a 1:1 mixture of methyl esters.

Thus chlorination of the anhydropenicillin in the presence of addedchloride ions causes a slight improvement in the 2'S: 2'R ratio.

Attempted Reaction of2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoic acidwith sodium azide.

The 2'R acid (50 mg., 0.14 mmole) was added to a solution of sodiumazide (10 mg., 0.15 mmole) in water (10 ml.). Complete dissolution wasachieved upon addition of a few drops of 5% bicarbonate solution. Thesolution was heated on the steam bath for 4 hours, by which time it wasdark orange in color. After cooling, the solution was brought to pH 4with N HCl and extracted with methylene chloride. The extract was washedwith water, dried over anhydrous magnesium sulfate and evaporated toyield 24 mg. of material. The IR spectrum of this material showed noβ-lactam absorption at 5.6μ .

In a second experiment, a solution of sodium azide (20.5 mg., 0.33mmole) in dimethylformamide (30 ml.) was prepared by brief warming onthe steam bath, and the 2'R acid (100 mg., 0.29 mmole) was added. Theresulting solution was heated on the steam bath for 19 hours, thencooled, diluted with water, brought to pH 4 with N HCl and extractedwith methylene chloride. After washing with water and drying overanhydrous magnesium sulfate the methylene chloride was evaporated to aresidue of 47 mg. A small amount of crystalline material was obtainedupon treatment of this residue with methylene chloride-petroleum ether.The IR spectrum of this material showed no azide peak at 4.7μ and onlyphthalimido absorption at 5.62μ. Evaporation of the mother liquor gave aresidue whose IR spectrum showed a weak azide peak at 4.72μ and someβ-lactam absorption at 5.60μ .

Conversion of methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoateinto methyl2-(2'S-azido-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoate.

Sodium azide (10.1 mg., 0.16 mmole) was dissolved in DMF (15 ml.) bybrief warming on the steam bath, and to this solution was added themethyl ester having the 2'R-chloro configuration (50 mg., 0.14 mmole).The resulting solution was stirred at room temperature for 24 hours andthen diluted with water and extracted with methylene chloride. Themethylene chloride extract was washed thoroughly with water, dried overanhydrous magnesium sulfate and evaporated to dryness. The resultingwhite crystalline solid was recrystallized from chloroform-petroleumether to give 42 mg. of unreacted 2'R ester.

In a second experiment the reaction mixture was heated on the steam bathfor 5 hours, by which time the color had become a deep yellowish-orange.Isolation of the product as described above afforded 38 mg. of a solidresidue whose IR spectrum showed azide absorption at 4.7μ and carbonylpeaks at 5.58, 5.62, 5.79 and 5.82μ. The NMR spectrum showed, in theβ-lactam region, 1-proton absorptions at 4.22 and 4.71 (d,J=2.0).

In a third experiment, sodium azide (20.2 mg., 0.31 mmole) was dissolvedin 10 ml. of freshly distilled DMF, the 2'R ester (100 mg., 0.28 mmole)was added, and the solution was heated 5 hours on the steam bath. Theproduct was isolated in the usual manner and was crystallized from2-butanone-ethanol to give 25 mg. of 2'S-azido ester (first crop), m.p.142°-144° .

Anal. Calcd. for C₁₇ H₁₅ N₅ O₅ : C, 55.29; H, 4.09; N, 18.97 Found: C,55.45; H, 4.02; N, 18.68.

The NMR spectrum shows peaks at 2.22 (4H,d), 4.22 (1H,d,J=2.0 Hz), 4.71(1H,d,J=2.0 Hz), 6.17 (3H), 7.70 (3H), 7.91 (3H).

The structure of the compound is ##STR48##

In a further experiment sodium azide (20.2 mg., 0.28 mmole) wasdissolved in freshly distilled DMF (10 ml.) by heating on the steam bathfor 0.5 hours. Then the 2'R-chloro ester (100 mg., 0.28 mmole) wasadded, followed by an additional 20.2 mg. of sodium azide. The resultingmixture was heated at 90° for 3 hours, with stirring. Isolation asdescribed above yielded a semi-solid residue which weighed 68.2 mg.(67%) and whose NMR spectrum was identical to that described above.Chromatography on a 1×2 column of alumina (Woelm, grade II) and elutionwith methylene chloride afforded 62 mg. of crystalline material.Recrystallization from methylene chloride-petroleum ether afforded 38mg. in the first crop, m.p. 144°-145° (colorless prisms).

Conversion of methyl2-(2'S-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoateinto methyl2-(2'R-azido-3'-S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoate.

The pure 2'S-chloro ester (100 mg., 0.28 mmole) was reacted with sodiumazide (2 molar equivalents) in DMF (10 ml.) as in the last experimentdescribed above. Isolation in the same manner afforded 52.2 mg. ofmaterial. This material was chromatographed on a 4.5 = 1 cm column ofalumina (Woelm, activity II). Elution with methylene chloride affordedno material in the first 10 ml. followed by 13.0 and 11.1 mg. ofcrystalline material in each of the next 10 ml. Both residues had thesame NMR spectrum. They were therefore combined and recrystallized fromchloroform-petroleum ether to give 19 mg. of colorless needles, m.p.183°-187° dec.

The NMR spectrum shows peaks at 2.10 (4H,d), 4.18 (1H,d,4.0 Hz), 4.33(1H,d,4.0 Hz), 6.10 (3H), 7.47 (6H). Anal. Calcd. for C₁₇ H₁₅ N₅ O₅ : C,55.29; H, 4.09; N, 18.97. Found: C, 55.06; H, 4.25; N, 19.10.

The structure of this compound is ##STR49##

Chlorination of the p-toluenesulfonic acid salt ofanhydro-6-aminopenicillin.

The salt (328 mg., 0.89 mmole) was suspended in methylene chloride (30ml.), the mixture cooled to 0°, and gaseous chlorine was introduced for15 seconds. Dry nitrogen was then passed into the resulting yellowsolution for 15 minutes to remove excess chlorine, and the solution wasevaporated to dryness. Trituration of the residue with dry etherafforded a quantitative yield of the mixture of two compounds shownbelow: ##STR50##

The mixture was a stable (below 20°) white powder, m.p. 148°-149° dec.The IR spectrum showed peaks at 5.55 and 5.62 μ. The 2'R-isomer (formedin 80% yield) showed NMR absorption CDCl.sub. 3) at τ 2.20 (2H,d), 2.80(2H,d) 4.02 (1H,d,4.2Hz), 4.82 (1H,d,4.2 Hz), 7.63 (3H), 7.80 (3H), 8.00(3H). (The NH₂ protons were not observed.) The 2'S-isomer (formed in 20%yield) showed NMR absorption at 2.20 (2H,d), 2.80 (2H,d), 4.82 (2H,m),7.63 (3H), 7.83 (3H), 8.00 (3H).

Chlorination of anhydro-6-tritylamino penicillin, the precursor of thep-toluenesulfonic acid salt, was unsuccessful using Cl.sub. 2, SO₂Cl.sub. 2, pyrrolidone hydrotrichloride, pyridinium trichloride,PCl.sub. 5, and (C₆ H₅ O)₃ PCl. In each case a reaction of the desiredtype occurred, but the trityl group was then lost and the productdecomposed.

Chlorination of the p-toluenesulfonic acid salt in the presence ofexcess tetraethylammonium chloride did not alter the ratio of the 2'Rand 2'S isomers.

Reaction of the chlorination product of the p-toluenesulfonic acid saltof anhydro-6-aminopenicillin with methanol.

The anhydropenicillin salt (3.80 g., 10.3 mmoles) was suspended inmethylene chloride (150 ml.), the mixture was cooled to -5°, and a slowstream of dry nitrogen was passed through it. Gaseous chlorine waspassed into the mixture for 30 seconds, and the nitrogen stream was thencontinued for 15 min. Methanol (75 ml.) was added and the resultingyellow solution was swept with nitrogen at 0° for 20 min. The coolingbath was then removed and, after 15 min., the solvent was removed underreduced pressure at 25°. The residue was treated with 5 ml. of methylenechloride and the resulting solution re-evaporated; this procedure wasrepeated three times and at this point the pale yellow foam had no odorof chlorine or HCl. The foam was dissolved in dry acetone, the orangesolution was filtered to remove a small amount of insoluble material,and petroleum ether (30°-60°) was added to the cloud point. A few dropsof acetone were then added to clarify the solution, and crystallizationwas allowed to proceed at 10° C. The white crystals thus obtained werewashed with cold 1:1 acetone-petroleum ether, and dried in vacuo. Theyield was 2.7 g. (67%), m.p. 125°-130° dec. The compound has thestructure shown ##STR51##

Anal. Calcd. for C₁₆ H₂₁ N₂ O₆ Cl: C, 47.48; H, 5.23. Found: C, 47.62;H, 5.54.

The NMR spectrum shows peaks at 2.22 (2H,d), 2.90 (2H,d), 4.02 (1H,d,4.0Hz), 4.87 (1H,d,4.0 Hz), 6.48 (3H), 7.65 (3H), 7.78 (3H), 8.15 (3H).

Interconversion of the methyl esters of the2'R-chloro-3'-S-aminotosylate and 2'R-chloro-3'-S-phthalimido series.

The crude methyl ester obtained by chlorination of 193 mg. (0.48 mmole)of the p-toluenesulfonic acid salt of anhydro-6-aminopenicillin, andthen treatment with methanol, was dissolved in methylene chloride. Thesolution was cooled to 0° C and shaken with an ice-cold 5% sodiumbicarbonate solution. The colorless organic layer was dried overanhydrous magnesium sulfate, the volume reduced to 5 ml., andN-carboethoxyphthalimide (100 mg., 0.46 mmole) was added. The reactionmixture was allowed to stand overnight at room temperature and thesolvent was then removed. The residue was chromatographed on a column ofWoelm alumina (neutral, grade II). Elution with 1:1 benzene-methylenechloride afforded 37 mg. of undefined material followed by 68 mg. (afterrecrystallization form chloroform-petroleum ether) of the methyl esterof 2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoicacid, identical in all respects with the compound obtained fromanhydro-6-phthalimidopenicillin.

The reaction sequence just described is: ##STR52##

Hydrolysis of the chlorination product of the p-toluenesulfonic acidsalt of anhydro-6-aminopenicillin

The salt (200 mg.) was chlorinated in the usual way and, after removalof the solvent, the residue was maintained under high vacuum for 30 min.It was then dissolved in a mixture of D₂ O (2 ml.) and CD₃ COCD₃ (2ml.), cooled to 0°, and the NMR spectrum was recorded at intervals.Hydrolysis was complete after 3 hr., and the solvent was, therefore,removed by lyophilization. The resulting foam was redissolved in D₂ O.The NMR spectrum of this solution indicated that quantitative conversionto a 4:1 mixture of 2'R:2'S chloro acids had been achieved. ##STR53##

The 2'R-chloro acid showed NMR absorptions at 2.37 (2H,d), 2.78 (2H,d),3.78 (1H,d,4.0 Hz), 4.93 (1H,d,4.0 Hz), 7.77 (3H), 7.88 (3H), 8.09 (3H).The epimeric 2'S-chloro acid had NMR absorption at 2.37 (2H,d), 2.78(2H,d), 4.53 (1H,d,2.0 Hz), 4.57 (1H,d,2.0 Hz), 7.77 (3H), 7.98 (3H),8.24 (3H).

Methyl ester of the2-(2'R-chloro-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoic acid.

The methyl ester of the 2'R-chloro-3'S-aminotosylate was neutralizedwith bicarbonate, as already described. Evaporation of the methylenechloride solution gave the free base, a crystalline compound, m.p.67°-70° dec. The compound was not stable in the solid state and darkenedperceptibly after 10 minutes at room temperature.

The NMR spectrum of the compound has peaks at 3.97 (1H,d, 4.1 Hz), 5.38(1H,d,4.1 Hz), 6.22 (3H), 7.70 (3H), 8.00 (3H). The position of the NH₂protons varies with time; although these peaks shift, no other changesoccur in the spectrum. The IR spectrum of the compound has peaks at2.94, 5.60, 5.80, 6.12μ which (in CH₂ Cl.sub. 2) do not change withtime.

The structure of this compound is ##STR54## Methyl ester of2-(2'S-chloro-3'-S-amino-4'-oxo)azetidinyl-3-methyl-2-bu

The 2'R-chloro-3'-S-aminotosylate (440 mg., 1.09 mmole) andtetramethylguanidinium chloride (1.387 g., 9.1 mmoles, 9 molarequivalents) were refluxed in spectroscopic grade chloroform (9 ml.) for4.5 hours. The solution was then cooled to 0°, washed with ice-cold 5%bicarbonate solution, dried over anhydrous magnesium sulfate, andevaporated to dryness under reduced pressure at 25°. The NMR spectrum ofthe residue indicated it to be a 5:1 mixture of the 2'S- and2'R-epimers, the predominant isomer being the 2'S compound, whosestructure is: ##STR55## The NMR spectrum of this compound shows peaks at4.45 (1H,d,1.8 Hz), 5.63 (1H,d,1.8 Hz), 6.18 (3H), 7.70 (3H), 8.00 (3H).The position of the NH₂ protons depends upon the age of the solution.

Complete separation of the two epimers could be achieved by aluminachromatography and elution with 1:1 benzene-ethyl acetate. However,because of the instability of these compounds it was usually moreconvenient to separate at a later stage.

Conversion of the p-toluenesulfonic acid salt of methyl2-(2'R-chloro-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoate to the2'S-azido-3'S-amino ester.

The crystalline methyl ester having the 2'R-chloro-3'S-TsOH.H₂ N--configuration (448 mg., 1.1 mmoles) and tetramethylguanidinium azide(347 mg., 2.2 mmoles, 2 molar equivalents) were refluxed for 75 minutesin 20 ml. of anhydrous chloroform. The solution was allowed to cool andexcess anhydrous ether was added to precipitate tetramethylguanidiniumchloride, which was removed by filtration. The resulting material, afterremoval of the solvent, was the desired 2'S-azide-3'S-amino ester (237gm., 90%) which appeared pure by t.l.c. and NMR. The NMR spectrum hadpeaks at 4.97 (lH, d, 1.8 Hz), 5.87 (lH,d,1.8 Hz), 6.20 (3H), 7.00 (2H),7.72 (3H), 8.02 (3H).

The compound has the structure ##STR56##

The compound could also be isolated by washing the chloroform reactionmixture with cold water, drying and evaporating. Tetraethylammoniumazide (in various solvents) and sodium azide (in DMF) were lesseffective for this reaction.

Conversion of the p-toluenesulfonic acid salt of methyl2-(2'S-chloro-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoate to the2'R-azido-3'S-amino methyl ester.

A 4:1 mixture of 2'S- and 2'R-chloro-3'S-amino esters from anepimerization of the 2'R-chloro compound (532 mg., 2.29 mmoles), andtetramethylguanidinium azide (500 mg., 3.2 mmoles) were refluxed in purecloroform (10 ml.) for 4.5 hours. The mixture was then washed withice-cold 5% bicarbonate solution, dried over anhydrous magnesiumsulfate, and evaporated to give 465 mg. (85%) of a 3.3:1 mixture of2'R-and 2'S-azido-3'S-amino methyl esters. This mixture was separated bychromatography on neutral alumina (Woelm, activity II). Elution withbenzene:ethyl acetate (2:3) afforded the pure 2'R-azido-3'S-amino estersas a crystalline compound. Recrystallization from chloroform-petroleumether gave long needles, m.p. 116°-117°.

Anal. calc'd for C₉ H₁₃ N₅ O₃ : C, 45.19; H, 5.48; N, 29.28. Found: C,45.42; H, 5.24; N, 28.81.

The IR spectrum has peaks at 4.72, 5.62 and 5.80μ. The NMR spectrumshows peaks at 3.97 (1H,d,4.1 Hz), 4.38 (d,4.1 Hz), 6.22 (3H), 7.60(2H), 7.70 (3H), 8.00 (3H).

The compound has the structure ##STR57##

Separation of the 2'S- and 2'R-azido-3'S-amino methyl esters via thep-toluenesulfonic acid salts.

A 1:1 mixture of the 2'S- and 2'R-azido compounds (239 mg.) wasdissolved in dry acetone (3 ml.) and the solution was treated with anequivalent amount of p-toluenesulfonic acid hydrate. Crystallizationbegan after 5 min. After 15 hr., acetone (1 ml.) was added and, after anadditional 20 min., ether (5 ml.). Filtration then gave 150 mg. (72%recovery, based on one isomer) of a crystalline p-toluenesulfonic acidsalt. Regeneration of the free base (NaHCO₃ -H₂ O-CH₂ Cl₂) revealed thatthis was the salt of the 2'S-azido compound. The separation procedure isthus ##STR58##

Phenylacetylation of Methyl2-(2'R-chloro-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoate.

The free 2'R-chloro-3'S-amino ester, obtained from 722 mg. (1.78 mmoles)of the p-toluenesulfonic acid salt, was dissolved in methylene chloride(15 ml.), and phenylacetic acid (242 mg., 1.78 mmoles) was added. Thissolution was treated, dropwise with stirring, with a solution ofdicyclohexylcarbodiimide (405 mg., 1.96 mmoles). Precipitation ofdicyclohexylurea commenced before the addition was complete. Thereaction mixture was allowed to stand at room temperature for threehours, by which time no free amine remained (by t.l.c.). Most of theurea was then removed by filtration, and the filtrate was washed withice-cold 5% bicarbonate solution. After drying over anhydrous magnesiumsulfate, the organic phase was evaporated to give a solid residue. Thiswas dissolved in boiling chloroform and an equal volume of petroleumether (30°-60°) was added, whereupon crystallization occurred. The crudeproduct weighed 700 mg. One more recrystallization from the same solventmixture gave 500 mg. (80%) of methyl2-(2'R-chloro-3'S-phenylacetamido-4'-oxo)azetidinyl-3-methyl-2-butenoate, m.p. 111°-115° dec. The NMR spectrum of this compound showspeak at 2.62 (5H), 3.10 (1H,d,10 Hz), 3.94 (1H,d, 4.2 Hz), 4.34(1H,q,4.2,10 Hz), 6.22 (3H), 6.31 (2H), 7.70 (3H), 8.02 (3H). The samecompound could be obtained, but in slightly lower yield usingdiisopropylcarbodiimide or phenylacetyl chloride and triethylamine. Thecompound has the structure ##STR59##

Phenylacetylation of Methyl2-(2'S-chloro-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoateCyclization to Oxazoline.

The p-toluenesulfonic acid salt of methyl2-(2'R-chloro-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoate (651 mg.,1.6 mmoles) and tetramethylguanidinium chloride (1.210 g., 5 molarequivalents) were refluxed in spectroscopic grade chloroform (9 ml.) for4.5 hours. The resulting solution was cooled, washed successively withice-cold 5% bicarbonate, ice-cold saturated sodium chloride, dried overanhydrous magnesium sulfate, and then used immediately for the next step(previous experiments had indicated that the above procedure leads to a5:1 or 6:1 excess of the 2'S-chloro-3'S-amino compound over the2'R-chloro-3'S-amino compound). To the solution were added phenylaceticacid (288 mg., 1.7 mmoles), diisopropylcarbodiimide (222 mg., 1.8mmoles) and methylene chloride (20 ml.), and this solution was refluxedfor 1 hour. Removal of the solvent gave a residue whose NMR spectrumshowed peaks at 2.73 (phenyl), 4.72 (d, 1.9 Hz), 5.33 (d, 1.9 Hz), 6.30(OCH₃), 6.47 (CH₂), 7.80 (CH₃), 8.05 (CH₃). The structure of thiscompound is ##STR60##

The spectrum also showed peaks at 4.02 (d,3.8 Hz), 4.80 (d, 3.8 Hz),6.33, 7.83, 8.43. This is the spectrum of the compound having thestructure, ##STR61## i.e.,2-benzyl-6(1'-methoxycarbonyl-2'-methylprop-1'-enyl)-1-oxa-3,6-diaza-4S,5R-bicyclo[3,2,0]hept-2-en-7-one,which is formed by intramolecular cyclization of the preceedingcompound. The cyclization occurs on standing, but is facilitated byheating of a solution of the chloro compound, by shaking withbicarbonate, or by chromatography on alumina or silica gel of eitherepimeric 2'-chloro-3'-acylamino ester, as will be described below.

Chromatography of the above mixture on silica gel and elution withpetroleum ether - ethyl acetate (1:1) afforded the oxazoline.Recrystallization from ethyl acetate gave 240 mg. (49% from thep-toluenesulfonic acid salt), m.p. 126.5°-127°.

The NMR spectrum of the pure compound shows peaks at 2.73 (5H), 4.02(1H,d,3.8 Hz), 4.80 (1H,d,3.8 Hz), 6.30 (3H), 6.33 (2H), 7.83 (3H), 8.43(3H).

Formation of the oxazoline upon chromatography of thecis-2'R-chloro-2'S-phenylacetamido methyl ester.

The crystalline cis compound (100 mg.) was chromatographed on alumina(Woelm, grade II). Elution with carbon tetrachloridebenzene (1:1) gave38 mg. of the oxazoline in the first fractions, followed by uncyclizedcis compound.

In a second experiment, the cis compound (1.0 g.) was chromatographed on37 g. of silica gel, elution being performed with 1:1 petroleum ether(30°-60°): ethyl acetate. There were obtained in successive fractions450 mg. of recovered cis compound, 140 of a mixture of the cis compoundand oxazoline, and 210 mg. of oxazoline. The transformation is ##STR62##

In a third experiment the crystalline 2'R-chloro-3'S-amino methyl ester(157 mg., 0.68 mmole), in dry methylene chloride (8 ml.), was treatedsuccessively with phenylacetic acid(92 mg., 0.68 mmole) anddiisopropylcarbodiimide (93 mg., 0.74 mmole). The solution was stirredat room temperature for 22 hours, and the precipitated diisopropylureawas then collected by filtration. Addition of ether to the mother liquorcompleted the precipitation of the urea. Evaporation of the filtrate,followed by chromatography on a 1.0×7 cm column of alumina (activity II)gave, with 1:1 benzene-methylene chloride, 83 mg. of crystallinematerial. This was found, by NMR and t.l.c. to be a mixture of oxazolineand 2'R-chloro-3'S-phenylacetamido ester.

Isolation of Methyl2-(2'R-phenylacetoxy-3'S-phenylacetamido-4'-oxo)azetidinyl-3-methyl-2-butenoateand oxazoline following phenylacetylation of the epimeric methyl2-(2'-chloro-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoate (4.428 g.,10.9 mmoles) and 8.260 g. (54.5 mmoles) of freshly preparedtetramethylguanidinium chloride were refluxed for 2 hours inspectroscopic grade chloroform (65 ml.). The cooled solution was washedsuccessively with ice-cold 5% bicarbonate solution, ice-cold saturatedsodium chloride solution, and then dried over anhydrous magnesiumsulfate. Then a solution of phenylacetic acid (1.490 g., 11 mmoles) anddiisopropylcarbodiimide (1.500 g., 11.9 mmoles) in methylene chloride(25 ml.) was added, and the mixture was refluxed for 2 hours. The NMRspectrum of the reaction product showed only a small amount of theoxazoline (based on the peaks at 7.83 and 8.43). The solution was thenwashed with ice-cold 5% bicarbonate, dried over anhydrous magnesiumsulfate, and evaporated. The NMR spectrum of this residue now showedoxazoline as the major product. Thus, washing with bicarbonate hadcaused cyclization. The product was left at 10° overnight, thenredissolved in methylene chloride and shaken again with bicarbonatesolution. After drying and evaporation of the organic layer the NMRspectrum of the residue was redetermined; it now indicated that themixture contained 70% of oxazoline. The mixture, in 1:1 ethylacetate:petroleum ether, was filtered through 50 g. of silica gel andthen chromatographed carefully on 110 g. of silica gel. Elution wasperformed with graded mixtures of petroleum ether and ethyl acetate (250ml. of 20% ethyl acetate, followed by 200 ml. of 30% ethyl acetate,followed by 200 ml. of 30% ethyl acetate, followed by 200 ml. of 35%ethyl acetate, followed by 200 ml. of 45% ethyl acetate, followed by 50%ethyl acetate); 40 ml. fractions were collected. Fractions 1-3 afforded100 mg. of methyl2-(2'R-chloro-3'S-phenylacetamido-4'-oxo)azetidinyl-3-methyl-2-butenoate. Fractions 5-12 afforded 1.475 g. of oxazoline. Fractions13-15 were rechromatographed and yielded, besides an additional 150 mg.of oxazoline (total 1.625 g., 48%), 240 mg. of a compound assigned thestructure ##STR63## This compound has IR absorption at 2.9, 3.0, 5.60,5.70, 5.80, 5.98, 6.05, 6.65μ. The NMR spectrum has peaks at 2.65-2.75(10H,m), 3.26 (1H,d,9.5 Hz), 3.68 (1H,d,4.1 Hz), 4.53 (q,4.1, 9.5 Hz),6.27 (3H), 6.43 (2H), 6.54 (2H), 7.82 (3H), 8.28 (3H). The mass spectrumshows a molecular ion at m/e 450. Phenylacetylation of methyl2-(2'R-azido-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoate.

The crystalline amine azide (415 mg., 1.74 mmole) was dissolved in drymethylene chloride (10 ml.) and to this solution were added,successively, phenylacetic acid (235 mg., 1.73 mmole) anddiispropylcarbodiimide (220 mg., 1.75 mmole). The solution was refluxedfor 9 hours, and the solvent was then removed. The residue wastriturated with cold carbon tetrachloride, and the carbontetrachloride-soluble material (now free of most of the dissopropylurea)was chromatographed an alumina (Woelm, grade II). Elution withbenzene-ethyl acetate afforded 641 mg. of pale yellow oil. This wasrechromatographed to yield 601 mg. (97%) of methyl2-(2'R-azido-3'S-phenylacetamido-4'-oxo)azetidinyl-3-methyl-2-butenoateas long needles, after recrystallization from carbontetrachloride-petroleum ether; m.p. 102°-103°. The compound has thestructure ##STR64##

anal. Calcd. for C₁₇ H₁₉ N₅ O₄ : C, 57.13; H, 5.36; N, 19.60. Found: C,65.76; H, 5.35; N, 19.42. The NMR spectrum shows peaks at 2.72 (5H),3.38 (1H,br), 4.57 (1H,d,4.2 Hz), 4.72 (1H,d,4.2 Hz), 6.27 (3H), 6.38(2H), 7.77 (3H), 8.05 (3H).

Phenylacetylation of methyl2-(2'S-azido-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoate

The amino azide (323 mg., 1.35 mmole) was dissolved in methylenechloride (10 ml.) containing triethylamine (110 mg., 1.09 mmole), andphenylacetylchloride (170 mg., 1.1 mmole), in methylene chloride (5ml.), was added dropwise with stirring. After 30 min., the reactionmixture was washed with five 10 ml. portions of water, dried overanhydrous magnesium sulfate, and evaporated. Chromatography on alumina(Woelm, activity II) and elution with benzene afforded 185 mg. of methyl2-(2'S-azido-3'S-phenylacetamido-4'-oxo)azetidinyl-3-methyl-2-butenoateas a colorless oil. The compound has the structure: ##STR65##

The NMR spectrum has peaks at 2.67 (5H), 3.22 (1H,d,8 Hz), 4.73(1H,d,2.0 Hz), 5.32 (1H,q,2.0,8 Hz), 6.25 (3H), 6.38 (2H), 7.73 (3H),8.00 (3H).

The same compound was obtained in 83% yield using phenylacetic acid anddiisopropylcarbodiimide in methylene chloride.

Conversion of Methyl2-(2'R-chloro-3'S-phenylacetamido-4'-oxo)azetidinyl-3-methyl-2-butenoateinto methyl2-(2'S-azido-3'S-phenylacetamido-4'-oxo)azetidinyl-3-methyl-2-butenoate.

Tetramethylguanidinium azide (126 mg., 0.8 mmole), in spectroscopicgrade chloroform (3 ml.), was treated dropwise with stirring with asolution of the chloride (229 mg., 0.66 mmole) in chloroform (5 ml.).When the addition was complete, the reaction mixture was refluxed fortwo hours. At this time no unreacted chloride remained. The reactionsolution was washed with water, dried over anhydrous magnesium sulfateand evaporated. The residue was chromatographed on silica gel to obtain75 mg. of the azide, identical with that described in the preceedingexperiment.

The equation for this reaction is: ##STR66##

Preparation of t-butyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoate andt-butyl2-(2'S-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoate byreaction of the anhydro-6-phthalimidopenicillin chlorination productwith t-butanol.

A 2:1 mixture of2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoylchloride and its 2'S epimer was stirred overnight at room temperature inmethylene chloride containing five molar equivalents of t-butanol.Evaporation of the solution then yielded a mixture containing only 15%of t-butyl esters (based on integration of the t-butoxy peak at 8.47 inthe n.m.r. spectrum). The conversion increased to 40% when the mixedacid chlorides were refluxed overnight in methylene chloride-t-butanol.Refluxing for 20 hours in t-butanol solvent effected a 75% conversion toa mixture of y-butyl esters containing the same 2:1 ratio of epimers asthe acid chloride precursor. Chromatography on silica gel separatedthese compounds. The cis-isomer was obtained as a colorless oil; itsn.m.r. spectrum showed peaks at 2.15 (4H), 3.85 (1H, d, 4 Hz), 4.38 (1H,d, 4), 7.71 (6H), 8.46 (9H).

The compound has the structure ##STR67##

The trans-isomer crystallized on standing. Recrystallization fromethanol gave material melting at 170°-172°. The n.m.r. spectrum showspeaks at 2.29 (4H, d), 3.85 (1H, d, 2.0 Hz), 4.55 (1H, d, 2.0), 7.73(3H), 1.98 (3H), 8.47 (9H).

Anal. Calcd. for C₂₀ H₂₁ N₂ O₅ Cl: C, 59.32; H, 5.23; N, 6.91. Found: C,59.05; H, 5.36; N, 6.75.

This compound has the structure ##STR68##

Reaction of t-butyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoatewith trifluoroacetic acid.

The ester (100 mg.) was dissolved in trifluoroacetic acid (2 ml.). Aftertwo minutes at room temperature the solvent was removed under reducedpressure. The residue was recrystallized from chloroform-petroleum etherto give2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoicacid, identical in all respects to the compound prepared by directhydrolysis of the dichloride.

Allylic Bromination of Methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3- methyl-2-butenoate.

To a suspension of the methyl ester (100 mg., 0.276 mmole) in carbontetrachloride (7.5 ml.) were added N-bromosuccinimide (108 mg., 0.61mmole) and benzoyl peroxide (2 mg.). When this mixture was brought toreflux, the ester dissolved; the heat source was then replaced by a 100watt Photoflood No. 2 lamp and reflector mounted close to the reactionflask and stirring was continued until the reaction was complete (10min.). The resulting suspension was cooled, diluted with a small amountof chloroform, and the precipitated succinimide was removed byfiltration. Evaporation of the filtrate gave a white crystalline solidresidue whose n.m.r. spectrum showed no absorption in the region of theallylic methyl groups (7.5 to 8.0), but did show succinimide absorptionat 7.2. This was removed, in part, by trituration with carbontetrachloride and methylene chloride, and the total product was thenchromatographed on neutral alumina (Woelm, activity II). Elution withmethylene chloride afforded a crystalline dibromo compound which, afterrecrystallization from carbon tetrachloride-petroleum ether, melted at65°-70° dec. and weighed 116 mg. (80%).

The compound has the structure ##STR69##

Anal. Calcd. for C₁₇ H₃₁ N₂ O₅ ClBr₂ : C, 39.22; H, 2.52; N, 5.38.Found: C, 38.95; H, 2.36; N, 5.40.

The n.m.r. spectrum has peaks at 2.18 (4H), 3.80 (1H, d, 4.2 Hz), 4.21(1H, d, 4.2 Hz), 5.07 (1H, d, 10.2 Hz), 5.23 (1H, d, 10.9 Hz), 5.27 (1H,d, 10.2 Hz), 5.33 (1H, d, 10.9 Hz), 6.11 (3H). The spectrum appearsunusually complex because one of the --CH₂ Br groups is cis to themethoxycarbonyl substituent and the other is trans; and, in each --CH₂Br group, the methylene protons are magnetically non-equivalent. Thei.r. spectrum has peaks at 5.55, 5.62 and 5.79 μ.

Allylic bromination of methyl2-(2'S-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoate.

The compound (100 mg.) was reacted with two molar-equivalents ofN-bromosuccinimide in exactly the same manner as described above.Chromatography on alumina (Woelm, activity II) and elution withmethylene chloride afforded 122 mg. (84%) of the desired compound as aclear viscous oil which appeared homogeneous by n.m.r. and t.l.c. Then.m.r. spectrum shows peaks at 2.08 (4H), 3.62 (1H, d, 1.7 Hz), 4.27(1H, d, 1.7 Hz), 5.05 (1H, d, 10.0 Hz), 5.36 (1H, d, 10.0 Hz), 5.38 (1H,d, 11.0 Hz), 5.57 (1H, d, 11.0 Hz), 6.04 (3H).

This compound has the structure ##STR70##

Allylic bromination of methyl2-(2'R-azido-3'S-phenylacetamido-4'-oxo)azetidinyl-3-methyl-2-butenoate.

The methyl ester (136 mg., 0.38 mmole), N-bromosuccinimide (210 mg.,1.18 mmole, 3-molar-equivalents), and benzoyl peroxide (2 mg.) wereheated at reflux in spectroscopic grade carbon tetrachloride; the heatsource was then removed and replaced with a 100 watt Photoflood No. 2lamp mounted close to the reaction vessel. The illumination/refluxingwas terminated after 9 minutes. The resulting suspension was cooled,filtered, and the reddish filtrate washed with ice-cold 10% bicarbonatesolution. The resulting yellow solution was dried over anhydrousmagnesium sulfate and evaporated to give 143 mg. (77%) of a viscous oil.Chromatography on alumina and elution with benzene, followed bychloroform afforded 29 mg. of material, having the same n.m.r. spectrumas the reaction product, which did not crystallize. This n.m.r. spectrumshows peaks at 2.67 (5H,m), 3.33 (1H, br), 4.32 (1H, d, 4.0 Hz), 4.50(1H, d, 4.0 Hz), 5.38 (2H, sl.br.), 5.63 (2 H, sl.br.), 6.17 (3H), 6.18(2H). The i.r. spectrum has peaks at 2.97, 4.71, 5.60 5.75 and 5.95 μ.The spectra are those of a compound having the structure ##STR71##

Reaction of methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoatewith one molar equivalent of N-bromosuccinimide.

A suspension of the cis compound (75 mg., 0.207 mmole),N-bromosuccinimide (36.9 mg., 0.207 mmole) and benzoyl peroxide (1.5mg.) in carbon tetrachloride (5 ml.) was heated to reflux until a clearsolution resulted. The heat source was then removed and the mixture wasstirred while being illuminated with a 100 watt Photoflood No. 2 amp.After 10 minutes the reaction was terminated and the mixture was cooledto room temperature and filtered to give succinimide (17 mg., 85% of thetheoretical amount), identified by its melting point and infraredspectrum. The filtrate was evaporated to dryness to give a white solidresidue which, on the basis of its NMR spectrum contained a small amountof succinimide and an exactly 1:1 mixture of the two possiblemonobrominated compounds. The solid was chromatographed on alumina toremove the succinimide. Elution with methylene chloride afforded the 1:1mixture of monobrominated compounds as a white foam (87.5 mg., 96%). TheNMR spectrum of this mixture shows peaks at 2.20 ( 4H), 3.83 (0.5H,d,4.23.87 (0.5H,d,4.2 Hz), 4.28 (0.5H,d,4.2 Hz), 4.32 (0.5H,d,4.2 Hz),5.10-5.88 (2H,m,overlapping AB quartets), 6.15 (1.5H), 6.17 (1.5H), 7.55(3H).

The structures of these compounds are ##STR72## methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-bromo-trans-2-butenoateand methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-bromo-cis-2-butenoate.

Conversion of the cis- and trans-methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-bromo-2-butenoatesto the cis- and trans-methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-azido-2-butenoates.

The 1:1 mixture of monobrominated compounds (87.5 mg., 0.198 mmole), inspectroscopic grade chloroform (3 ml.), was treated withtetramethylguanidinium azide (34.4 mg., 0.218 mmole). A pale yellowcolor appeared immediately. The solution was stirred at room temperaturefor 3 hours (under these conditions the 2'-chloro substituent is stable)and was then a pale yellow-brown in color. It was diluted withchloroform, washed with water, decolorized with activated carbon anddried over anhydrous magnesium sulfate. evaporation of the solventafforded 83.9 mg. of a white solid residue. Examination of this residueby t.l.c. showed no unreacted monobrominated compounds. The IR spectrumhad peaks at 4.70 (azide), 5.52 (β-lactam), 5.58 and 5.79μ(phthalimido), 5.75 (ester) and 6.16μ. The NMR spectrum showed that theazides had been obtained. It had peaks at 2.17 (4H), 3.78 (1H,d,4.0 Hz),4.22 (1H,d,4.0 Hz), 5.12-5.87 (2H, overlapping AB quartets), 6.12 (3H),7.58 (3H). The two isomers apparently have the same NMR spectrum. Thestructures of these compounds are ##STR73## methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-azido-trans-2-butenoateand ##STR74## methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-azido-cis-2-butenoate.

Conversion of the cis- and trans- methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-azido-2-butenoatesto the cis- and trans-methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-amino-2-butenoates.

The mixture of azides (83.9 mg.) in benzene (10 ml.), was hydrogenatedover Adams catalyst (50 mg.) at 45 psi for 3 hours. The reactionsolution was then diluted with chloroform and passed through a pad ofanhydrous magnesium sulfate. Evaporation at 25° afforded a sticky solidresidue. The IR spectrum showed a peak at 4.7 μ about half the intensityof one in the same position in the starting material. The NMR spectrumshowed peaks due to the azide and, as well, some new peaks. Reductionwas, therefore, incomplete. The hydrogenation was repeated for anadditional 3 hours at 45 p.s.i. The resulting mixture was diluted withchloroform and passed through a pad of anhydrous magnesium sulfate. Theblack filtrate was not decolorized upon filtration through Celite orupon making (of an aliquot) with 5% sodium bicarbonate solution.Evaporation at room temperature afforded a black solid residue (92 mg.)whose IR spectrum showed complete disappearance of the azide absorptionand whose NMR spectrum showed disappearance of the multiplets in the 5-6τ region and appearance of new multiplets in the 7τ region. The solidwas redissolved in chloroform, treated with activated carbon and passedthrough a pad of anhydrous magnesium sulfate to give a clear colorlesssolution. Evaporation gave 87 mg. of a solid residue. The NMR spectrumshows peaks at 2.18 (4H), 3.88 (1H,d,4.1 Hz), 4.33 (1H,d,4.1 Hz),6.90-7.20 (2H,m), 6.20 (3H), 7.67 (1.5H), 7.70 (1.5H). The structures ofthe amino compounds are ##STR75## methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-amino-trans-2-butenoateand ##STR76## methyl2-(2'R-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-amino-cis-2-butenoate.

Cyclization of methyl2-(2'R-chloro-3'S-phthalimido-4-oxo)azetidinyl-3-methyl-4-amino-trans-2-butenoateto 3-methyl-4-methoxycarbonyl-7S-phthalimido-1,5-diaza-6S-bicyclo[4,2,0] oct-3-en-8-one.

The above mixture of amines are recovered unchanged after standing for16 hours at room temperature and after 3 hours refluxing in chloroform.

The mixed amino esters (60.5 mg., 0.159 mmole), in anhydrous t-butanol(10 ml.), were treated with freshly-prepared potassium t-butoxide (17.8mg., 0.159 mmole). The initially pale yellow solution immediately turneddark brown. It was stirred at room temperature for 1 hour and thenpoured into ice-cold saturated ammonium chloride solution and extractedwith chloroform. The chloroform extract was washed with water, driedover anhydrous magnesium sulfate, decolorized with activated carbon, andevaporated at 25° to give 60.7 mg. of a solid residue. This waschromatographed on a 0.5×4.5 cm column of 1.0 g. of neutral alumina(Woelm, activity II), Flution with methylene chloride affored 40 mg. ofsolid in the first 5 ml. This was rechromatographed on a 0.5×5.5 cmcolumn of 1.2 g. of alumina. Flution with 5 ml. of 1:1 benzenemethylenechloride gave 28 mg. of solid material whose IR spectrum showed onlyweak absorption at 5.6μ. Flution with 10 ml. of methylene chloride thengave 8.6 mg. of a crystalline compound, m.p. 121°-122° . The IR spectrumof this compound (KBr) showed peaks at 2.92 (N-H), 5.56 (β-lactam),5.62, 5.79 (phthalimido), 5.82 (ester) and 6.03μ (C=C). The NMR spectrumhad peaks at 2.25 (4H), 3.87 (1H,d,2.0 Hz), 4.30 (1H,d,2.0 Hz), 6.23(3H), 7.70 (2H), 7.88 (3H). ##STR77## The systematic name for thenucleus is ##STR78## 1,5-diaza-6S-bicyclo [4,3,0] octan-8-one.

Reaction of methyl2-(2'S-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoatewith one molar equivalent of N-bromosuccinimide

A mixture of the pure trans methyl ester (165 mg., 0.455 mmole),N-bromosuccinimide (81 mg., 0.455 mmole) and benzoyl peroxide (4 mg.),in carbon tetrachloride (12 ml), was brought to reflux. The heat sourcewas then removed and replaced with a 100 watt Photoflood No. 2 lamp.Illumination of the stirred reaction mixture was maintained for 15 mins.The suspension was then cooled, filtered, and the filtrate evaporated toa white foam. This was chromatographed on a 0.5 × 13 cm column of 3.0 gof neutral alumina (Woelm, activity II). Elution with methylene chlorideafforded 206 mg. of a 60:40 mixture of mono bromo compounds. The n.m.r.spectrum of this mixture has peaks at 2.17 (4H), 3.73 (1H, d, 2Hz), 4.37(1H, d, 2Hz), 5.27- 5.65 (2H, m, two overlapping AB quartets), 6.15(3H), 7.59 (1.2H), 7.82 (1.8H).

The structures of these compounds are ##STR79## methyl2-(2'S-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-bromo-trans-2-butenoateand ##STR80## methyl2-(2'S-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-bromo-cis-2-butenoate.

Conversion of the cis- and trans-methyl2-(2'S-chloro-3'S-phthalimido-4'oxo)azetidinyl-3-methyl-4-bromo-2-butenoatesto the cis- and trans-methyl2-(2'S-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-azido-2-butenoates.

The 60:40 mixture of monobrominated compounds (205 mg., 0.455 mmole), inchloroform (4 ml), was treated with tetramethylquanidinium azide (79.1mg., 0.50 mmole). The solution immediately turned light yellow. It wasstirred at room temperature for 4 hrs. and then diluted with chloroform,washed with water, treated with activated carbon, and dried overanhydrous magnesium sulfate. Evaporation then afforded 184 mg. of themixed azides; i.r.: 4.7, 5.55, 5.62, 5.79μ; n.m.r.: 2.17 (4H), 3.76 (1H,m, overlapping doublets), 4.40 (1H, d, 2.0 Hz), 5.52 and 5.58 (0.8H, ABquartet of one isomer), 5.87 (1.2H, br), 6.15 (3H), 7.67 (1.2H), 7.87(1.8H).

The structures of these compounds are ##STR81## methyl2-(2'S-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-azido-trans-2-butenoate##STR82## methyl2-(2'S-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-4-azido-cis-2-butenoate

Reaction of2-benzyl-6-(1'-methoxycarbonyl-2'-methylprop-1'-enyl)-1-oxo-3,6-diaza-4S,5R-bicyclo [3,2,0] hept-2-en-7-one with one molar equivalent of alithium thioalkoxide in hexamethylphosphoric triamide

(Hydrolysis of the oxazoline ester to the oxazoline acid andrearrangement to the oxygen analog of Penicillin G

The equation of the above-mentioned reaction is ##STR83##

For the various experiments described below, HMPT was purified bydistillation, under dry nitrogen, from lithium hydride or lithiumaluminum hydride at 116°-117° C and 19-20 torr. It was stored overmolecular sieves in a flask fitted with a rubber septum, and removed bysyringe when needed. The lithium reagent was generated in HMPT by one ofthe following methods (Method A and Method B):

Method A:

A round-bottomed flask was equipped with a magnetic stirrer and rubberseptum. Two syringes were inserted into the septum, and dry nitrogen waspassed through the flask. Then HMPT was introduced and the nitrogensweep was continued for 1 hr. The desired amount of mercaptan was thenadded followed, at 25° , by an equivalent amount of a solution ofn-butyllithium in hexane. The nitrogen sweep was continued for 15 min.and the flask was then sealed and stored in the refrigerator, aliquotsof the reagent being withdrawn for reaction as needed. The solution wasstable for about one month.

Method B:

Lithium hydride was placed in a round-bottomed flask equipped withmagnetic stirrer and rubber septum. The flask was flushed with nitrogenand HMPT was then added. The nitrogen sweep was then continued for 30min. before addition of the mercaptan. The resulting mixture was stirredfor 1.5 hr. at room temperature and was then filtered (under nitrogen ina dry box). The concentration of the reagent was determined by titrationof an aliquot with N NCl, and the flask was then sealed and stored inthe refrigerator until needed.

Experiment 1

The oxazoline (200 mg., 0.64 mmole), in HMPT (1.5 ml.) was treated,under nitrogen and with magnetic stirring, with 1.3 ml. of a 0.58 Msolution (0.75 mmole) of t-BuSLi in HMPT prepared by method A. Theaddition was performed at room temperature during 0.5 hour and thereaction mixture was then allowed to stir overnight. Ice-cold water andether were then added and the pH was adjusted to 2.3. The ether extractwas washed with cold water, dried over anhydrous magnesium sulfate andevaporated to give a yellow oil. This was dissolved in ether and thesolution extracted with ice-cold 5% bicarbonate. The bicarbonate extractwas brought to pH 3 and extracted with ether. Evaporation of the driedether extract gave 120 mg. of yellow oil which showed β-lactamabsorption in the IR at 5-6μ and in the NMR at 4.8-5.0τ. This NMRspectrum changed after 7 hours at room temperature.

Experiment 2

The oxazoline (62 mg., 0.197 mmole), in HMPT (1 ml.), was swept withnitrogen for 30 min., and 0.35 ml. of a 0.615 M solution of t-BuSli(0.215 mmole), prepared by method A, was then added dropwise during 2hr. The mixture was stirred for 2 hr. after addition was complete andthe acidic material was then isolated as described in experiment 1. Itwas a yellow oil weighting 23 mg.

Experiment 3

The oxazoline (66 mg., 0.21 mmole), in HMPT (3 ml.), was swept withnitrogen, and 0.3 ml. of a 0.73 M solution of n-C₃ H₇ Li, prepared bymethod A, was then added dropwise in two portions; 0.2 ml. were addedduring 0.5 hr., the mixture was stirred for 1.5 hr., and the remainderwas then added during 0.5 hr. The reaction mixture was kept at roomtemperature for 8 hr. and then stored overnight in the refrigerator.Isolation yielded 24 mg. of acidic material.

Experiment 4

The oxazoline (64 mg., 0.20 mmole) was dissolved in HMPT (1 ml.) and thesolution was degassed with a stream of dry nitrogen for 45 min. Then0.44 ml. of a 0.47 M solution of t-BuSLi (prepared by method B) wasadded during 35 min. Stirring was continued for 2 hr. and the acidicmaterial was then isolated. It weighted 15 mg.

Each of the acidic compounds isolated from experiments 2-4 featured NMRpeaks at 2.52 (phenyl), 3.77 (d,3.5 Hz), 4.0 (d,3.5 Hz), 6.18, 7.77 and8.47. These are the peaks of a compound having the structure ##STR84##which could be obtained in crystalline form by an alternative route.Each of the NMR spectra of the acids from experiments 2-4 also showedextraneous peaks, the most prominent being a singlet at τ 6.3 and asinglet at 8.75, corresponding to a proton alpha to a carboxylic acidand methyl groups attached to a quaternary centre.

The crystalline oxazoline carboxylic acid was found to be inactive as anantibacterial agent at levels of 250 mcg./ml. However, all of the acidicfractions prepared by hydrolysis of the methyl ester showed significantantibacterial activity. The acid from experiment 3 (which also containedthe biologically inactive oxazoline carboxylic acid) was dissolved indimethylsulfoxide, chromatographed, and compared with Penicillin G. Thetwo substances were spotted on duplicate 1/2 inch strips of S and S 589blue ribbon paper. After overnight development in a system containingn-butanol 60:acetic acid:15:water 25, the strips were air dried and oneset was sprayed with Riker penicillinase. Both sets were then subjectedto bioautography on B. subtilis. The antibacterial agent in the acidfrom experiment 3 was found to have an Rf virtually identical to that ofpenicillin G and the bioactivity was eliminated by penicillinasetreatment, as expected for a compound having a structure similar to thatof penicillin G.

MIC data on penicillin G and on the acid from experiment 3 aresummarized in the following table.

    __________________________________________________________________________                  Penicillin G Control                                                                      Acid from Experiment 3                              Organism      MIC  mcg./ml.                                                                             M.I.C.  mcg./ml.                                    __________________________________________________________________________    D. pneumoniae .004        8                                                   + 5% serum*                                                                   Str. pyogenes .004        8                                                   + 5% serum*                                                                   S. aureus Smith                                                                             .016        16                                                  at 10.sup.-.sup.4 dil'n                                                       S. aureus Smith                                                                             .06         63                                                  at 10.sup.-.sup.4 dil'n                                                       + 50% serum                                                                   S. aureus BX-1633-2                                                                         125         63                                                  at 10.sup.-.sup.3 dil'n                                                       S. aureus BX-1633-2                                                                         >125        125                                                 at 10.sup.-.sup.2 dil'n                                                       S. aureus Meth.                                                                             125         32                                                  Resist. at 10.sup.-.sup.3 dil'n                                               S. aureus at 10.sup.-.sup.3 dil'n                                                           63          32                                                  S. aureus at 10.sup.-.sup.2 dil'n                                                           >125        >125                                                Sal. enteritidis at                                                                         0.13        63                                                  10.sup.-.sup.4 dil'n                                                          E. coli Juhl at 10.sup.-.sup.4                                                              32          63                                                  dil'n                                                                         E. coli at 10.sup.-.sup.4 dil'n                                                             125         125                                                 K. pneumoniae at 10.sup.-.sup.4                                                             2           63                                                  dil'n                                                                         Pr. mirabilis at 10.sup.-.sup.4                                                             1           63                                                  dil'n                                                                         Pr. morganii at 10.sup.-.sup.4                                                              >125        63                                                  dil'n                                                                         Ps. aeruginosa at 10.sup.-.sup.4                                                            >125        63                                                  dil'n                                                                         Ser. marcescens at 10.sup.-.sup.4                                                           >125        63                                                  dil'n                                                                         __________________________________________________________________________     *45% AAB at 10.sup.-.sup.3 dil'n                                              + 50% Medium Shown                                                       

On the basis of its antibacterial spectrum, susceptibility topenicillinase, and NMR spectrum the antibacterial agent prepared duringthe hydrolysis of the oxazoline ester is assigned the structure##STR85##2,2-dimethyl-3R-carboxy-6S-phenylacetamido-1-oxo-4-aza-5R-bicyclo[3,2,0] heptan-7one.

Reaction of2-benzyl-6-(1-'methoxycarbonyl-2'-methylprop-1'-enyl)-1-oxa-3,6-diaza-4S,5R-bicyclo[3,2,0] hept-2-en-7-one with two equivalents of a lithium thioalkoxidein hexamethylphosphoric triamide.

The oxazoline (74 mg., 0.236 mmole) was dissolved in HMPT (1.5 ml.) andthe solution was degassed with dry nitrogen for 0.5 hr. Then 0.83 ml. ofa 0.612 M solution of t-BuSLi (0.508 mmole), prepared by method A, wasadded during 0.5 hr. and the mixture was stirred overnight. The acidicfraction weighed 33 mg. and showed antibacterial activity in a plateassay vs. S. lutea. The NMR spectrum showed β-lactam protons at 4.0 and4.7 Hz, phenyl absorption at 2.6, methylene absorption at 6.12, asinglet at 6.2 and methyl peaks at 8.75. The characteristic peak of theoxazolinecarboxylic acid,2-benzyl-6-(1'-carboxy-2'-methyl-prop-1'-enyl)-1-oxa-3,6-diaza-4S,5R-bicyclo[3,2,0] hept-2-ene-7-one, were absent from the spectrum.

The same result was obtained from an experiment with t-BuSLi prepared bymethod B.

These experiments demonstrate that hydrolysis of2-benzyl-6-(4'-methoxycarbonyl-2'-methylprop-1'-methylprop-1'-enyl)-1-oxa-3,6-diaza-4S,5R-bicyclo [3,2,0] hept-2-en-7-one to the carboxylic acid is effected bylithium thioalkoxides in HMPT and that rearrangement to theoxapenicillin occurs under the experimental conditions.

Reaction of the epimeric methyl2-(2'-chloro-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoates withbenzyl chloroformate (carbobenzoxy chloride).

The crystalline p-toluenesulfonic acid salt of methyl2-(2'R-chloro-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoate (576 mg.,1.42 mmoles) was dissolved in spectroscopic grade chloroform (6 ml.),and 1.080 g. (5 molar equivalents) of tetramethylguanidinium chloridewere added. The mixture was refluxed for 3.5 hr. and then cooled, washedwith water, dried over anydrous magnesium sulfate, and carbobenzoxychloride (297 mg., 1.7 mmoles) was added followed, dropwise, by asolution of triethylamine (185 mg., 1.83 mmoles) in dry chloroform (5ml.). After 45 min. stirring at room temperature the reaction wasstopped by addition of water. The organic layer was dried overevaporated to a residue to 353 mg. This was chromatographed on silicagel and the column eluted with 1:1 petroleum ether (30°-60°) -- ethylacetate; 17 thirty ml. fractions were collected. Fractions 2-4 gave 108mg. of a compound having the structure ##STR86## The NMR spectrum ofthis compound has peaks at 2.67 (5H), 4.20 (1H,d,2Hz), 4.87 (2H), 5.35(1H,d,2Hz), 6.27 (3H), 7.73 (3H), 8.03 (3H).

Fractions 12-17 afforded 54 mg. of a compound assigned the structure##STR87## This fused β-lactam-oxazolidone has a molecular ion at m/e 240in its mass spectrum. The IR spectrum has peaks at 3.0, 5.56, 5.62,5.78, 6.10μ. The NMR spectrum has peaks at 2.87 (1H,br), 3.92(1H,d,4Hz), 5.08 (1H,d,4Hz), 6.20 (3H), 7.70 (3H), 8.02 (3H).

Reaction of 2-(2'-chloro-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoicacid with carbobenzoxy chloride. ##STR88##

The p-toluenesulfonic acid salt of anhydrio-6-amino-penicillin (557 mg.,1.51 mmoles) was chlorinated in methylene chloride at 0°. The solventwas then removed under reduced pressure at 20°. The residue wasdissolved in a mixture of water (4 ml.) and acetone (5 ml.) and thissolution was maintained at 0° for 3 hr. to effect hydrolysis of the acidchloride. Then sodium bicarbonate (705 mg., 8.4 mmoles) was addedfollowed, dropwise at 0°, by a solution of carbobenzoxy chloride (660mg., 1.51 mmoles) in dry acetone (5 ml.). The reaction mixture wasstirred for 1 hr. and the product was then isolated by dilution withwater, extraction with ether, drying over anhydrous magnesium sulfate,and evaporation. The IR spectrum showed peaks at 3.0, 5.60, 5.62,5.82-5.88 μ.

This experiment demonstrates that the amino acid can be acylatedsuccessfully.

Chlorination of anhydro-α-phenoxyethylpenicillin

The anhydropenicillin (500 mg., an 80:20 L:D mixture of side chaindiastereomers) was chlorinated at room temperature in methylene chlorideusing a strong sweep of chlorine for 15-20 seconds. The solvent was thenremoved at 20° . The resulting product is a mixture of isomers havingthe structure ##STR89## Its NMR spectrum shows peaks at 2.3 (1H,d,9Hz),2.75 (2H,d, 9Hz), 3.13 (2H,d,9Hz), 3.92 (1H,m), 4.45 (1H,m), 5.28(1H,m), 7.74 (3H), 7.85 (3H), 8.40 (3H,d,7Hz). The IR spectrum showspeaks at 2.95, 5.55, 5.91μ.

This experiment demonstrates that a 6-acylamino anhydropenicillin can bechlorinated successfully.

Hydrolysis of the anhydro-α-phenoxyethylpenicillin chlorination product.

The above-mentioned trichloro compound was dissolved in acetone (5 ml.),the solution cooled to 0° , and a solution of sodium bicarbonate (284mg.) in ice-cold water (5 ml.) was added dropwise with stirring. Someoily material precipitated during this addition and acetone (15 ml.)was, therefore, when the addition was complete. The resulting paleyellow solution was stirred for 2 hr. at 0° and then diluted with coldwater (100 ml.). Extraction with methylene chloride, drying overanhydrous magnesium sulfate, and evaporation afforded a pale yellow oil.This material is assigned the structure ##STR90## The IR spectrum showdpeaks at 2.9, 5.58, 5,85μ. The compound was dissolved in methylenechloride and the solution extracted with 5% sodium bicarbonate. Theaqueous extract was re-acidified to pH 2.5 and re-extracted withmethylene chloride. Evaporation of the dried methylene chloride extractgave material, having high antibacterial activity vs. S. lutea, which isassigned the structure ##STR91## Treatment of this material withdiazomethane gave a methyl ester which showed, in the IR, a peak at5.6μand, in the NMR a peak at 8.75 consistent with the presence of aβ-lactam and methyl groups attached to an oxazolidine ring.

In a second experiment, the chlorination product from 215 mg. (0.62mmole) of anhydroα-phenoxyethylpenicillin was dissolved in ice-coldtetrahydrofuran (3 ml.) and to the colorless solution was added ice-coldwater (2 ml.). The resulting solution was stirred at 0° for 2 hr., thendiluted with ice-cold saturated sodium chloride and extracted withmethylene chloride. Evaporation of the dried methylene chloride extractafforded 277 mg. of a white foam. The NMR spectrum of this dichloro acidshows peaks at 2.30 (1H, br), 2.72 (2H, d, 8Hz), 3.10 (2H, d, 8Hz), 3.83(1H, br), 4.40 (1H, br), 5.22 (1H,q), 7.67 (3H), 7.93 (3H), 8.40 (3H,d). The IR spectrum showed peaks at 2.96, 5.60, 5.8 μ.

Reaction of 2-(2'-chloro-3'S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoicacid with phenylacetyl chloride. ##STR92##

The p-toluenesulfonic acid salt of anhydro-6-aminopenicillin (242 mg.)was chlorinated and the product hydrolyzed in aqueous acetone as alreadydescribed. To the resulting solution (volume 20 ml.) at 0° was added inportions sodium bicarbonate (275 mg.) followed by a solution ofphenylacetyl chloride (152 mg.) in acetone (5 ml.) in one portion. Themixture was stirred for 30 min. and then diluted with water andextracted with methylene chloride; this extract was discarded. Theaqueous phase was brought to pH 3 and extracted with methylene chloride.This extract was dried over anhydrous magnesium sulfate and evaporatedto give the desired compound. The IR and NMR spectra were consistentwith the assigned structure.

Chlorination of anhydrobenzylpenicillin

Anhydropenicillin G (176 mg., 0.56 mmole) was dissolved in methylenechloride (7 ml.) and the solution was chlorinated at room temperaturefor 30 sec. with a strong sweep of chlorine. The solvent was thenremoved immediately at 20° under reduced pressure to give a pale yellowfoam. The IR spectrum of this foam shows peaks at 2.92, 5.5, 5.95μ. TheNMR spectrum shows that the product is a 4:1 mixture of2-(2'-R-chloro-3'S-phenylacetamido-4'-oxo)azetidinyl-3-methyl-2-butenoylchloride and its 2'S epimer. The major isomer has the structure##STR93## Its NMR spectrum shows peaks at 2.71 (5H), 3.07 (1H, d, 9Hz),3.97 (1H, d, 4.5 Hz), 4.52 (1H, q, 4.5, 9Hz), 6.37 (2H), 7.78 (3H), 7.92(3H).

The minor isomer has the structure ##STR94## Its NMR spectrum showspeaks at 2.71 (5H), 3.07 (1H, d, 9Hz), 4.12 (1H, d, 2Hz), 5.17 (1H, q,2,9 Hz), 6.44 (2H), 7.78 (6H).

Hydrolysis of the anhydrobenzylpenicillin chlorination product

The above-mentioned mixture of dichlorides (from 176 mg. ofanhydropenicillin) was dissolved in acetone (6 ml.) and water (4 ml.)was added followed, at 0°, by excess sodium bicarbonate. The mixture wasstirred for 2.3 hr. and then diluted with water (50 ml.) and extractedthrice with methylene chloride and once with either. Acidification ofthe aqueous phase to pH 2.5, extraction with methylene chloride andevaporation of the dried methylene chloride extract afforded 36 mg. ofan oil whose IR and NMR spectra indicated it to consist mainly ofunhydrolyzed acid chloride.

In a second experiment anhydropenicillin G (105 mg.) was chlorinated andthe resulting white foam was dissolved in ice-cold tetrahydrofuran (3ml.) and cold water (1 ml.) was added. The clear yellow solution wasmaintained at 0° for 2 hr. and then poured into a mixture of water andice, and extracted with methylene chloride. Evaporation of the driedmethylene chloride extract afforded 113 mg. (98%) of a chloro acid whoseIR and NMR spectra showed it to have the structure ##STR95##

The IR spectrum showed peaks at 3.05, 5.62, 5.90, 5.95μ. The NMRspectrum showed peaks at 2.72 (5H), 3.10 (1H, d, 8 Hz), 3.93 (1H, d, 4.5Hz), 4.42 (1H, q, 4.5, 8Hz), 6.32 (2H), 7.72 (3H), 8.01 (3H).

In a third experiment, the chloro acid from 333 mg. of anhydropenicillinG was treated in ether with an excess of ethereal diazomethane. Thesolution was stirred for 2 hr. at room temperature and then evaporatedto dryness. The residue was left in the refrigerator overnight and then,in methylene chloride, shaken with 5% bicarbonate, dried and evaporated.The residue was chromatographed on silica gel; elution with 1:1petroleum ether:ethyl acetate afforded 189 mg. (51%, m.p. 110.5°-111°)of the methyl ester having the structure ##STR96## and 40 mg. (12%) ofthe oxazoline ##STR97##

Both compounds were identical to those obtained from thep-toluenesulfonic acid salt of methyl2-(2'R-chloro-3'-S-amino-4'-oxo)azetidinyl-3-methyl-2-butenoate. Thereaction sequence proves that chlorination proceeds in the same mannerwith the phenylacetamido and amino side chains.

Synthesis of2-benzyl-6-(1'-carboxy-2'-methylprop-1'-enyl)-1-oxa-3,6-diaza-4S,5R-bicyclo[3.2.0] hept-2-en-7-one ##STR98##

The chloro acid prepared from 640 mg. of anhydropenicillin G wasdissolved in 5% bicarbonate solution. This was extracted with methylenechloride and then acidified to pH 2. Extraction with methylene chlorideand evaporation of the dried extract afforded a crystalline residue.Recrystallization from ethyl acetate gave 320 mg. (53%) of the oxazolinecarboxylic acid, m.p. 119°-122° dec. The IR spectrum shows a broad peakat 4μ, and as well peaks at 5.64, 5.92, 6.1μ, consistent with thezwitterionic structure ##STR99##

The NMR spectrum (in D₂ O) shows peaks at 2.67 (5H), 3.88 (1H, d,3.8Hz), 4.75 (1H, d, 3.8 Hz), 6.25 (2H), 8.09 (3H), 8.69 (3H).

Anal. Calcd. for C₁₆ H₁₆ N₂ O₄ : C, 63.99; H, 5.37; N. 9.33.

Found: C, 64.26; H, 4.89; N, 9.54.

Preparation of anhydrocarbobenzoxypenicillin

The p-toluenesulfonic acid salt of anhydro-6-aminopenicillin (2.90 g.,7.84 mmoles) was dissolved in methylene chloride (20 ml.) and thesolution was cooled to 0° and treated concurrently with pyridine (820mg.) in methylene chloride (15 ml.), and carbobenzoxy chloride (1.400g., 8.1 mmoles) in methylene chloride (15 ml.). Addition of the tworeagents was complete in 10 min. and, after an additional 10 min., themixture was washed with ice-cold N HCl, ice-cold saturated sodiumchloride, dried and evaporated to give 2.740 g. of a white foam. Thiswas crystallized from a mixture of ether and petroleum ether to give2.33 g. (89.5%) of anhydrocarbobenzoxypenicillin having the structure##STR100##

The NMR spectrum showed peaks at 2.65 (5H), 4.17 (1H), 4.45 (1H), 4.85(2H), 7.82 (3H), 7.93 (3H).

Preparation of anhydro-2,2',2"-trichloroethoxypenicillin

The p-toluenesulfonic acid salt of anhydro-6-aminopenicillin (2.396 g.,6.47 mmoles) was dissolved in methylene chloride (25 ml.) and thesolution was cooled and treated concurrently with pyridine (0.633 g., 8mmoles) in methylene chloride (10 ml.) and 2,2',2"-trichloroethylchloroformate (1.437 g., 6.9 mmoles) in methylene chloride (10 ml.). Atthe end of the addition t.l.c. examination of the reaction mixturerevealed a single spot (and a spot at the origin corresponding topyridinium chloride). The mixture was stirred an additional 15 min. andthen washed with cold N HCl, dried and evaporated. The IR and NMRspectra of the resulting white foam were consistent with the structure##STR101##

Conversion of anhydro-2,2',2"-trichloroethoxypenicillin into benzhydryl2-(2'R-chloro-3'S-trichloroethoxycarbonylamino-4'-oxo)azetidinyl-3-methyl-2-butenoate##STR102##

A. The anhydropenicillin (453 mg., 1.245 mmole) was chlorinated in theusual way to give a white foam. The NMR spectrum of this foam showedpeaks at 3.35 (1H, d, 10Hz), 3.83 (1H, d, 4.0Hz), 4.52 (1H, d,4.0,10Hz), 5.21 (2H), 7.71 (3H), 7.81 (3H).

B. Hydrolysis was performed at 0° for 2 hr. in tetrahydrofuran (10 ml.)and water (1 ml.). The resulting acid crystallized from chloroform. ItsNMR spectrum showed peaks at 3.17 (1H, d, 8Hz), 3.82 (1H, d, 4Hz), 4.56(1H, q, 4,8Hz), 5.20 (2H), 7.67 (3H), 7.93 (3H).

C. The acid was dissolved in hot benzene (40 ml.) and diphenyldiazomethane (363 mg., 1.87 mmoles) was added. The resulting solutionwas refluxed for 40 min. and then evaporated to dryness. The dark redoil was chromatographed on silica gel. Elution with graded mixtures ofpetroleum ether and ethyl afforded 480 mg. (70%) of the benzhydrylester. Its NMR spectrum showed peaks at 2.68 (10H), 3.06 (1H), 3.51 (1H,d, 9Hz), 4.04 (1H, d, 4.5Hz), 4.67 (1H, q, 4.5, 9Hz), 5.23 (2H), 7.72(3H), 7.98 (3H).

Equilibration of the R and S benzhydryl2-(2'-chloro-3'S-trichloroethoxycarbonylamino-4'-oxo)azetidinyl-3-methyl-2-butenoates

The benzhydryl ester (181 mg., 0.317 mmole) and tetramethylguanidiniumchloride (240 mg., 5 molar equivalents) were refluxed in chloroform (5ml.) for 5 hr. The cooled reaction mixture was then diluted withmethylene chloride, washed with saturated sodium chloride solution,dried and evaporated. The resulting white foam was found to be a 60:40mixture of the starting material and its 2'S epimer, which has thestructure ##STR103##

The NMR spectrum of this compound shows peaks at 2.68 (10H), 3.06 (1H),4.30 (1H, d, 2Hz), 4.95 (1H, d, 2Hz), 5.27 (2H), 7.72 (3H), 7.98 (3H).

In a second experiment, 4.851 g. (8.65 mmoles) of the 2'R isomer andtetramethylguanidinium chloride (7.9 g., 6-molar-equivalents) wererefluxed in spectroscopic grade chloroform (20 ml.) for 4 hr. Theproduct was a 70:30 mixture of 2'S:2'R isomers, i.e., these conditions,which involve a higher concentration of reactants, cause a sisgnificantimprovement in the proportion of the 2'S isomer.

Tetramethylguanidinium formate

Commercial 98% formic acid was distilled from anhydrous cupric sulfateat 46° and 113 torr. The distillate was redistilled from anhydrouscupric sulfate at 41° and 105 torr to give anhydrous formic acid.

Tetramethylguanidine (22 g., 0.19 mole) was dissolved in anhydrous ether(100 ml.) and to this solution at 0° was added a solution of anhydrousformic acid (7.1 ml., 0.198 mole) in anhydrous ether (40 ml.). Theresulting salt crystallized. It was collected, washed with ether, driedin high vacuum and recrystallized from chloroform-ether.

The salt was extremely hydroscopic and became oily after 3 min. exposureto atmospheric moisture. The oily material gave unsatifactory results inthe following experiments.

If the formic acid was not made anhydrous prior to formation of thesalt, the latter was obtained as an oil which gave unsatifactoryresults. The salt has the structure ##STR104##

Reaction of benzhydryl2-(2'-chloro-3'S-trichloroethoxycarbonylamino-4'-oxo)azetidinyl-3-methyl-2-butenoatewith tetramethylguanidinium formate.

Preparation of2-trichloroethoxy-6-(1'-benzhydryloxycarbonyl-2'-methylprop-1'-enyl)-1-oxa-3,6-diaza-4S,5R-bicyclo[3,2,0]hept-2-en-7-one

A 2:1 2'S:2'R mixture of benzhydryl esters (953 mg., 1.7 mmoles) andtetramethylguanidinium formate 4.992 g. (30.5 mmoles) were refluxed inspectroscopic grade chloroform (12 ml.). The reaction was complete in1.5 hr. and the mixture was, therefore, cooled, washed successively withwater and saturated sodium chloride, dried and evaporated. The residuewas chromatographed over 30 g. of silica gel. Elution was performed with100 ml. each of 90/10, 80/10 and 70/30 petroleum ether-ethyl acetate,with 30 ml. fractions being collected. Fractions 2-5 were combined togive 142 mg. (24%) of a compound having the structure ##STR105##

The IR spectrum of this oxazoline shows peaks at 5.61, 5.76, 5.81, 6.07μ.

The n.m.r. spectrum has peaks at 2.68 (10H), 3.08 (s, 1H), 3.99 (1H, d,3.5Hz), 4.86 (1H, d, 3.5Hz), 5.15 (1H, d, 12Hz), 5.27 (1H, d, 12Hz; themethylene protons of the trichloroethyl group are non-equivalent), 7.73(3H), 8.06 (3H).

The mass spectrum shows chlorine multiplets characteristic of thepresence of 3 chlorines, including a multiplet at m/e 522, the molecularion.

Chlorination of anhydrocarbobenzoxypenicillin

The anhydropenicillin (470 mg., 1.47 mmoles) was chlorinated inmethylene chloride for 15 seconds. After an additional 30 seconds thesolvent was removed to give a white foam which consisted of a 77:23mixture of dichlorides, the compound having the 2'R-chloro configurationpredominating. Its structure is ##STR106##

The NMR spectrum of this compound has peaks at 2.69 (5H), 3.14 (1H, d,9Hz), 3.93 (1H, d, 4Hz), 4.75 (1H, q, 4,9Hz), 4.83 (2H), 7.80 (3H), 7.90(3H)

The minor isomer has the structure ##STR107##

The NMR spectrum of this compound has peaks at 2.69 (5H), 3.14 (1H, d,9Hz), 4.10 (1H, d, 2Hz), 4.84 (2H), 5.17 (1H, d, 2Hz), 7.80 (6H).

Hydrolysis of the anhydrocarbobenzoxypenicillin chlorination product.

The dichloride (a 77:23 2'R:2'S mixture from 470 mg. of theanhydropenicillin) was dissolved in cold tetrahydrofuran (10 ml.) andice-cold water (1 ml.) was added. The mixture was left for 3 hr. at 0°and then added to ice-cold saturated sodium chloride solution.Extraction with methylene chloride and evaporation of the driedmethylene chloride extract afforded 535 mg. of the chloro acids. Themajor isomer has the structure ##STR108##

The NMR spectrum of this compound has peaks at 2.68 (5H), 3.63 (1H, d,10Hz), 3.87 (1H, d, 4Hz), 4.55 (1H, d, 4, 10Hz), 4.83 (2H), 7.72 (3H),7.98 (3H).

Reaction of2-(2'R-chloro-3'-S-benzyloxycarbonylamino-4'-oxo)azetidinyl-3-methyl-2-butenoicacid with sodium bicarbonate.

Preparation of the oxygen analog of a 5-epianhydropenicillin.

The chloro acid from 470 mg. of the anhydropenicillin, in acetone (5ml.), was added to a solution of sodium bicarbonate (247 mg., 2.94mmoles) in water (10 ml.). After 1 minute crystallization began. Themixture was stirred for 15 min. and then diluted with saturated sodiumchloride solution (20 ml.) and extracted twice with methylene chloride(10 ml. each) and once with ether (10 ml.). The combined organicextracts were dried over anhydrous magnesium sulfate and evaporated. Thecrystalline residue weighed 216 mg. Chromatography on silica gelafforded 185 mg. of material which was recrystallized to give acompound, m.p. 170°-172° dec., [α]_(D) ²⁵ -64.5° (c 0.45, CHCl₃), whichhas the structure ##STR109##

Anhydrocarbobenzoxypenicillin has [α]_(D) ²⁵ + 259° (c 1, CHCl₃). The5-epioxaanhydropenicillin has IR absorption at 2.98, 5.55, 5.65, 5.88and 6.51μ. The NMR spectrum has peaks at 2.44 (6H), 4.14 (1H, d, 1.8Hz),4.82 (1.8, 8H), 4.98 (1H, q, 1.8,8Hz), 7.82 (3H), 7.88 (3H). The massspectrum shows the molecular ion at 316.

Anal. Calcd. for C₁₆ H₁₆ N₂ O₅ : C, 60.75; H, 5.10; N, 8.86.

Found: C, 60.67; H, 5.20; N, 8.99.

Preparation of 6-phthalimido oxapenicillin

Anhydro-6-phthalimidopenicillin (606 mg.) was chlorinated in methylenechloride with a strong sweep of chlorine for 30 seconds and the solventwas then removed immediately. A 70:30 mixture of 2'S:2'R2-(2'chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoylchlorides was obtained. This mixture was hydrolyzed in a mixture oftetrahydrofuran (25 ml.) and water (2 ml.) at 0° for 4 hr. The resultingacid was a 77:23 mixture. The major isomer has the structure ##STR110##

Its NMR spectrum has peaks at 2.12 (4H), 3.62 (1H, d, 2Hz), 4.38 (1H, d,2Hz), 7.84 (3H), 8.11 (3H). The minor isomer, which has the 2'Rconfiguration, has already been described.

The trans acid was crystallized fro 1:1 ethyl acetate-ligroin, m.p.164°-166° dec. (sealed capillary). The crystalline trans acid inmethylene chloride, was shaken with 5 % bicarbonate for 5 min. Theaqueous phase was then separated, acidified to pH 4.5-5.0 and extractedwith methylene chloride. It was then acidified to pH 2.5 and extractedtwice with methylene chloride and twice with chloroform. Evaporation ofthe combined dried organic extracts afforded 30 mg. of material whichshowed antibacterial activity versus S. lutea at one-tenth the level ofphthalimidopenicillin. The NMR spectrum showed a peak at 8.73 for methylgroups on an oxazolidine ring and β-lactam absorption at 4.6. The activecompound has the structure ##STR111##

Preparation of benzhydryl2-(2'S-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoate.

The crystalline trans chloro acid (526 mg., 1.515 mmole) was dissolvedin boiling ethyl acetate (12 ml.) and the solution was then cooled toroom temperature and treated with diphenyldiazomethane (330 mg., a 10%excess) in ethyl acetate (18 ml.). Evolution of nitrogen commencedimmediately and was complete after 10 min. The mixture was then refluxedfor 1 hr. and evaporated to dryness. The residue was chromatographed on30 g. of silica gel. Elution with petroleum ether-ethyl acetate (70-30)removed some unreacted diphenyldiazomethane. Continued elution withpetroleum ether-ethyl acetate (1:1) afforded 772 mg. (99%) of thecompound having the structure ##STR112##

The n.m.r. spectrum shows peaks at 2.23 (4H, d), 2.67 (10H, m),3.00(1H,s), 3.87 (1H, d, 1.8Hz), 4.47 (1H, d, 1.8 Hz), 7.68 (3H), 7.90(3H).

Reaction of benzhydryl2-(2'S-chloro-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoatewith tetramethylguanidinium formate.

Preparation of benzhydryl2-(2'R-formyloxy-3'S-phthalimido-4'-oxo)azetidinyl-3-methyl-2-butenoate.

The benzhydryl ester (150 mg., 0.292 mmole) and tetramethylguanidiniumformate (260 mg., 1.61 mmoles, 5.5 molar equivalents) were refluxed inchloroform (spectroscopic grade) for 17 hr. The solution was washedsuccessively with water, saturated sodium chloride and water, and driedover anhydrous magnesium sulfate. The residue, after removal of thesolvent, was chromatographed over 18 g. of silica gel. Elution withgraded mixtures of petroleum ether-ethyl acetate afforded 54 mg. of acrystalline compound having the structure ##STR113##

The n.m.r. spectrum of this compound has peaks at -0.74(1H), 2.19(4H,d), 3.02(1H,s), 2.67(10H,m), 3.92 (1H, d, 3.8Hz), 4.59 (1H, d, 3.8Hz),7.75(3H), 8.11(3H).

Curtius rearrangement of the anhydrobenzylpenicillin chlorinationproduct.

Anhydropenicillin G (206 mg.) was chlorinated for 20 sec. at -6° C. inmethylene chloride. The solution was maintained for 2 min. at thistemperature and then evaporated to dryness. The residue, in chloroform(10 ml.), was treated at -6° C with tetramethylguanidinium azide (120mg., 1.2 molar equivalents) in chloroform (5 ml.). The resultingsolution was maintained for 1 hr. at -6° , and its IR spectrum was thendetermined. It showed peaks at 3.0, 4.67, 5.60, 5.95μ, as expected forthe formation of a compound having the structure ##STR114##

The acid azide was isolated by extracting the chloroform solution withwater, sodium chloride solution, drying, and removal of the solvent. Itwas then dissolved in methylene chloride (10 ml.), and the solutionheated to reflux. After 15 min. the IR spectrum showed a new peak at4.43μ, about twice as intense as the peak at 4.67μ. After an additional15 min. the peak at 4.43μ was now about six times as intense as the peakat 4.67μ. After a total of 2 hr. of refluxing the reaction seemedcomplete and the IR spectrum showed peaks at 3.12, 4.42, 5.58, 5.98μ.

Evaporation gave a compound having the structure ##STR115##

This isocyanate, in tetrahydrofuran (20 ml.), was added during 40 min.to 1:1 aqueous tetrahydrofuran containing 0.65 ml. of N HCl. The mixturewas stirred for 80 min. after addition was complete and was then dilutedwith water (200 ml.), saturated with sodium chloride and extractedexhaustively with methylene chloride. The methylene chloride extract wasdried over anhydrous magnesium sulfate and evaporated to give 139 mg. ofa white foam. The NMR spectrum of this foam showed β-lactam peaks in the3.9-4.7τ region, absence of peaks in the region of the allylic methylgroups and several peaks in the 8.5 -9τ region. Chromatography on silicagel afforded two compounds. One, a crystalline material, was identifiedas isobutyramide [(CH₃)₂ CHCONH₂ ]; the other was a 2:1 mixture ofepimers having the structure ##STR116##

The cis isomer, having the structure ##STR117## has NMR peaks at 2.63(5H), 3.5 (1H,d,8Hz), 3.94 (1H,d,5Hz), 4.32 (1H,q,8Hz), 6.28 (2H), 6.83(1H,m), 8.75 (6H,m).

The trans isomer, having the structure ##STR118## has NMR peaks at 2.63(5H), 3.5 (1H,d,8Hz), 4.07 (1H,d,2Hz), 5.48 (1H,q,2,8Hz), 6.33 (2H),6.83 (1H,m), 8.75 (6H,m).

The mass spectrum of the mixture of isomers shows an M-1 peak at 306 and308 and a peak at 272 corresponding to loss of chlorine from themolecular ion.

As disclosed above, the present invention includes a variety ofprocesses including those described in detail below. To avoidunnecessary repetition, it is to be noted that in the equations givenbelow there is frequent reference to the substituent having the formula##STR119## wherein frequently R² is hydrogen and R¹ is acyl. By acyl ismeant a group having the formula ##STR120## wherein R¹² represents2,2,2-trichloroethyl or benzyl; wherein R⁴ represents hydrogen, amino,carbobenzoxyamino, phenyl, fluoro, chloro, bromo, iodo, hydroxy,(lower)alkanoyloxy or (lower)alkoxy; X represents oxygen or sulfur; R⁵and R⁶ each represent hydrogen, phenyl, benzyl, phenethyl or(lower)alkyl; R⁷ represents (lower)alkyl; R⁸ and R⁹ each represent(lower)alkyl, (lower)alkylthio, benzylthio, cyclohexyl, cyclopentyl,cycloheptyl, benzyl, phenethyl, phenylpropyl, furyl, thienyl, naphthylor Ar; R¹⁰ represents (lower)alkylamino, di(lower)alkylamino,cycloalkylamino having 3 to 7 carbon atoms inclusive, allylamino,diallylamino, phenyl(lower)alkylamino, morpholino,lower(alkyl)morpholino, di(lower)alkylmorpholino,morpholino(lower)alkylamino, pyrrolidino, (lower)alkylpyrrolidino,di(lower)alkylpyrrolidino, N,N-hexamethyleneimino, piperidino,(lower)alkylpiperidino, di(lower)alkylpiperidino,1,2,5,6-tetrahydropyridino, N-(lower)alkylpiperazino,N-phenylpiperazino, N-(lower)alkyl(lower)alkylpiperazino,N-(lower)alkyl-di-(lower)alkylpiperazino, furfurylamino,tetrahydrofurfurylamino, N-(lower)alkyl-N-furfurylamino,N-alkyl-N-anilino or (lower)alkoxyanilino; Z¹, Z², and Z³ each represent(lower)alkyl or Ar--; R¹¹ represents (lower)alkyl, (lower)cycloalkyl,naphthyl, benzyl, phenethyl or ##STR121## and Ar represents a monovalentradical having the formula ##STR122## wherein R¹, R² and R³ are eachhydrogen, chloro, bromo, iodo, trifluoromethyl, phenyl, (lower)alkyl or(lower)alkoxy, but only one of said R¹, R² and R³ groups may representphenyl; and particularly hydrogen, hydrogen tosylate, phenylacetyl,phenoxyacetyl, carbobenzoxy, trichloroethoxycarbonyl,α-aminophenylacetyl, α-carbobenzyloxyaminophenylacetyl and, when R¹ andR² are taken in combination with the nitrogen atom to which they areattached, phthalimido. The same structures are also represented in someof the equations below as the acyl group ##STR123##

In some of the equations below esters of the carboxyl group arerepresented as ##STR124## in such cases R is (lower)alkyl, andpreferably methyl or t-butoxy, trichloroethyl, benzhydryl or benzyl.

With the above definitions in mind the preferred processes of thepresent invention are summarized as follows:

A. The process for producing a compound of the formula ##STR125##wherein R¹ is hydrogen or acyl and R² is hydrogen or, in combinationwith R¹ and the nitrogen atom to which they are attached, phthalimidowhich comprises chlorinating, [preferably wherein the chlorinating agentis chlorine, sulfuryl chloride or a complex of chlorine and pyridinehydrochloride], a compound of the formula ##STR126## where R¹ and R²have the meaning set out above;

B. The process of producing a compound of the formula ##STR127## whereinR¹ is hydrogen or acyl; R² is hydrogen or, in combination with R¹ andthe nitrogen atom to which they are attached, phthalimido; and R is(lower)alkyl, trichloroethyl, benzhydryl or benzyl which comprisesmixing with an alcohol having the formula ROH a compound of the formula##STR128## wherein R, R¹ and R² have the meaning set out above;

C. The process of producing a compound of the formula ##STR129## whereinR¹ is hydrogen or acyl and R² is hydrogen or, in combination with R¹ andthe nitrogen atom to which they are attached, phthalimido whichcomprises mixing with water a compound of the formula ##STR130## whereinR¹ and R² have the meaning set out above;

D. The process of producing a compound of the formula ##STR131## whereinR¹ is benzyl or phenoxymethyl and R is (lower)alkyl (and preferablymethyl or t-butyl), trichloroethyl, benzhydryl or benzyl which comprisesmixing with alumina or silica gel a compound of the formula ##STR132##wherein R and R¹ have the meaning set out above;

E. The process of producing a compound of the formula ##STR133## whereinR¹ is hydrogen or acyl; R² is hydrogen or, in combination with R¹ andthe nitrogen atom to which they are attached, phthalimido; and R is(lower)alkyl (and preferably methyl or t-butyl), trichloroethyl,benzhydryl or benzyl which comprises reacting with azide ion a compoundof the formula ##STR134## wherein R, R¹ and R² have the meaning set outabove;

F. The process of producing a compound of the formula ##STR135## whereinR¹ is hydrogen or acyl; R² is hydrogen or, in combination with R¹ andthe nitrogen atom to which they are attached, phthalimido; and R is(lower)alkyl (and preferably methyl or t-butyl), trichloroethyl,benzhydryl or benzyl which comprises reacting with about one mole of anallylic brominating agent (and preferably wherein the allylicbrominating agent is N-bromosuccinimide and a catalytic amount of anorganic peroxide is present) a compound of the formula ##STR136##wherein R, R¹ and R² have the meaning set out above;

G. The process of producing a compound of the formula ##STR137## whereinR¹ is hydrogen or acyl; R² is hydrogen or, in combination with R¹ andthe nitrogen atom to which they are attached, phthalimido, and R is(lower)alkyl (and preferably methyl or t-butyl), trichloroethyl,benzhydryl or benzyl which comprises reacting with about one mole ofazide ion (and preferably wherein the source of azide ion istetramethylguanidinium azide) a compound of the formula ##STR138##wherein R, R¹ and R² have the meaning set out above; H. The process ofproducing a compound of the formula ##STR139## wherein R¹ is hydrogen oracyl; R² is hydrogen or, in combination with R¹ and the nitrogen atom towhich they are attached, phthalimido; and R is (lower)alkyl,trichloroethyl, benzhydryl or benzyl which comprises hydrogenating(preferably in the presence of a platinum oxide catalyst a compound ofthe formula ##STR140## wherein R, R¹ and R² have the meaning set outabove;

I. The process of producing a compound of the formula ##STR141## whereinR¹ is benzyl or phenoxymethyl which comprises reacting with about onemole of a compound of the formula R² SLi wherein R² is (lower)alkyl (andpreferably n-propyl or t-butyl), in solution in the compound having theformula ##STR142## a compound of the formula ##STR143## wherein h₁ isbenzyl or phenoxymethyl and R is (lower)alkyl (and preferably methyl ort-butyl), trichloroethyl, benzhydryl or benzyl;

J. The process of producing the compound of the formula ##STR144## whichcomprises reacting with a deacylating agent a compound of the formula##STR145## wherein R¹ is benzyl or phenoxymethyl and the deacylatingagent is preferably S. lavendulae or E. coli or wherein the deacylationprocess is conducted by successive addition of trimethylchlorosilane,phosphorus pentachloride and methanol; K. The process of producing acompound of the formula ##STR146## wherein ##STR147## is acyl whichcomprises acylating the compound of the formula ##STR148## with acarboxylic acid of the formula R¹ -- COOH or its equivalent as anacylating agent for a primary amino group, and especially said processin which the acylating agent is the corresponding carboxylic acidchloride, acid bromide, and anhydride, acid mixed anhydride with a(lower)alkyl ester of carbonic acid or free acid in combination with acarbodiimide,

In another preferred embodiment of said process the acyl group##STR149## wherein B is an easily removable blocking group andespecially is selected from the group consisting of hydrochloric acid,carbobenzyloxy, aliphatic β-diketones and aliphatic β-diketo esters,e.g. hydrogen chloride or methyl acetoacetate;

L. The process of producing a compound of the formula ##STR150## wherein##STR151## is acyl which comprises acylating the compound of the formula##STR152## with a carboxylic acid of the formula R¹ -- COOH or itsequivalent as an acylating agent for a primary amino group, andespecially said process in which the acylating agent is thecorresponding carboxylic acid chloride, acid bromide, acid anhydride,acid mixed anhydride with a (lower)alkyl ester of carbonic acid or freeacid in combination with a carbodiimide,

In another preferred embodiment of said process the acyl group##STR153## wherein B is an easily removable blocking group andespecially is selected from the group consisting of hydrochloric acid,carbobenzyloxy, aliphatic β-diketones and aliphatic β-diketo esters,e.g. hydrogen chloride or methyl acetoacetate;

M. The process of producing the compound of the formula ##STR154## whichcomprises reacting with a deacylating agent a compound of the formula##STR155## wherein R¹ is benzyl or phenoxymethyl and the deacylatingagent is preferably S. lavendulae or E. coli or the deacylation processis conducted by successive addition of trimethylchlorosilane, phosphorusphentachloride and methanol; and

N. The process of producing a compound of the formula ##STR156## whereinR¹ is hydrogen or acyl and R² is hydrogen or, in combination with R¹ andthe nitrogen atom to which they are attached, phthalimio which comprisessaponifying, preferably with potassium t-butoxide a compound of theformula ##STR157## wherein R¹ and R² have the meaning set out above andR is (lower)alkyl, trichloroethyl, benzhydryl or benzyl.

The 4-chloroazetidine-2-ones of the present invention are usefulstarting materials for the preparation of the class of compounds calledsecopenicillins and described in detail in Belgium Pat. No. 754,125(Farmdoc 10051S); they are converted to secopenicillins by reaction withsulfur-containing compounds such as hydrogen sulfide, sodiumhydrosulfide, methyl mercaptan and benzylmercaptan or salts thereof(represented below as R⁶ S⁻).

Appropriate compounds of the present invention are converted to knowncephalosporins as follows: ##STR158## In two preferred embodiments X isBr and Y is H or Br.

There are thus provided alternative routes from penicillins produced bydirect fermentation, such as penicillins G and V, both to otherpenicillins and members of the cephalosporin family. As an example ofthe latter, where Y above is bromine, reaction with potassium5-methyl-1,3,4-thiadiazol-2-ylthiolate followed by conversion of R¹, R²and R to hydrogen (if they are not such already) provides the properly3-thiolated 7-ADCA nucleus which is then acylated in the usual mannerwith 1-tetrazolylacetic acid to produce cephazolin. As another exampleof the latter, when Y is hydrogen the product contains the nucleus7--ADCA (7-aminodesacetoxycephalosporanic acid). Thus when R² ishydrogen and R¹ is ##STR159## treatment with zinc and acetic acidproduces an ester of 7--ADCA which can be cleaved in the usual manner togive 7--ADCA itself.

As another example, cephalexin itself is produced by the same sequenceof reactions in which X is Br, Y is H and R¹ and R² taken together withthe nitrogen atom represent the group ##STR160## The anhydropenicillinused in that case as the original starting material is prepared eitherby treating N-nitrosohetacillin (prepared according to Belgium Pat. No.765,596, Farmdoc 67,311S) in the usual manner for converting apenicillin to an anhydro-penicillin or by coupling D-(-)-2-phenylglycine(as with a carbodiimide) with the compound of U.S. Pat. No. 3,311,638having the structure followed by formation of its acetone adduct andthen nitrosation according to Belgium Pat. No. 765,596, Fermdoc 67,511S.

Appropriate compounds of the present invention are used to preparepreviously known compounds in the penicillin series, includingpenicillins themselves, as illustrated by the following reactions:##STR161##

Cryptates have been described by B. Dietrich, J. M. Lehn and J. R.Saurage, J. Chem. Soc (D), 1055 (1970) and crown ether by C. J.Pedersen, J. Am. Chem. Soc., 89, 7017 (1967) and 92, 386, 391 (1970).Both cryptates and crown ether are agents which permit inorganic saltsto be dissolved in organic solvents by complexation.

Another procedure produces secopenicillins as follows: ##STR162## WhenR⁶ is benzyl it is converted to hydrogen by catalytic hydrogenation tofurnish another route to the sulfhydryl compounds above.

In the above equations -R³ is preferably ##STR163## and R¹, R², R³ andR⁴ have the meaning used previously herein.

In the treatment of bacterial infections in man, the new, bicyclicβ-lactam antibiotics of this invention, e.g. the oxapenicillins and theazacephalosporins are administered orally or parenterally, in accordancewith conventional procedures for antibiotic administration, in an amountof from about 5 to 200 mg./kg./day and preferably about 5 to20mg./kg./day in divided dosage, e.g., three to four times a day. Theare administered in dosage units containing, for example, 125 or 250 or500 mg. of active ingredient with suitable physiologically acceptablecarriers or excipients. The dosage units are in the form of liquidpreparations such as solutions or suspensions or as solids in tablets orcapsules.

The substituted azetidine-2-ones of the present invention are not activeas antibacterial agents but do function as inhibitors of the enzymeβ-lactamase and thus decrease the rate of destruction of a penicillinwhen used in combination therewith. This is demonstrated, for example,by reduction of the Minimum Inhibitory Concentration of ampicillinversus "resistant or β-lactamase producing bacteria such as P. morganiiand Ps. aeruginosa from values in the range of 500-1000 mcg./ml. tovalues as low as 125 mcg./ml. using concentrations of the substitutedazetidine-2-one in the range of about 125 mcg./ml. These particularfigures were obtained using the compound having the structure ##STR164##

I claim:
 1. A compound of the formulawherein ##STR165## is acyl otherthan phenylacetyl or phenoxyacetyl or a nontoxic, pharmaceuticallyacceptable salt thereof.
 2. A compound of claim 1 wherein ##STR166##wherein R¹² represents 2,2,2-trichloroethyl or benzyl; wherein R⁴represents hydrogen, amino, carbobenzoxyamino, phenyl, fluoro, chloro,bromo, iodo, hydroxy, (lower)alkanoyloxy or (lower)alkoxy where R₄ isnot H when Ar is phenyl; X represents oxygen or sulfur; R⁵ and R⁶ eachrepresent hydrogen, phenyl, benzyl, phenethyl or (lower)alkyl where R₅and R₆ are not both hydrogen where Ar is phenyl and X is oxygen; R⁷represents (lower)alkyl; R⁸ and R⁹ each represent (lower)alkyl,(lower)alkylthio, benzylthio, cyclohexyl, cyclopentyl, cycloheptyl,benzyl, phenethyl, phenylpropyl, furyl, thienyl, naphthyl or Ar; R¹⁰represents (lower)alkylamino, di(lower)alkylamino, cycloalkylaminohaving 3 to 7 carbon atoms inclusive, allylamino, diallylamino,phenyl(lower)alkylamino, morpholino, lower(alkyl)morpholino,di(lower)alkylmorpholino, morpholino(lower)alkylamino, pyrrolidino,(lower)alkylpyrrolidino, di(lower)alkylpyrrolidino,N,N-hexamethyleneimino, piperidino, (lower)alkylpiperidino,di(lower)alkylpiperidino, 1,2,5,6-tetrahydropyridino,N-(lower)alkylpiperazino, N-phenylpiperazino,N-(lower)alkyl(lower)alkylpiperazino,N-(lower)alkyl-di-(lower)alkylpiperazino, furfurylamino,tetrahydrofurfurylamino, N-(lower)alkyl-N-furfurylamino,N-alkyl-N-anilino or (lower)alkoxyanilino; Z¹, Z² and Z³ each represent(lower)alkyl or Ar--; R¹¹ represents (lower)alkyl, (lower)cycloalkyl,naphthyl, benzyl, phenethyl or ##STR167## and Ar represents a monovalentradical having the formula ##STR168## wherein R¹, R² and R³ and eachhydrogen, chloro, bromo, iodo, trifluoromethyl, phenyl (lower)alkyl or(lower)alkoxy, but only one of said R¹, R² and R³ groups may representphenyl.
 3. A compound of claim 1 wherein the acyl group ##STR169##wherein R represents (lower)alkyl.
 4. A compound of claim 1 wherein theacyl group ##STR170## wherein R¹ represents (lower)alkyl and R² and R³each represent a member selected from the group consisting of hydrogenand chloro.
 5. A compound of claim 1 wherein the acyl group ##STR171##wherein R¹ is (lower)alkyl and R² is a member selected from the groupconsisting of hydrogen and chloro.
 6. A compound of claim 1 wherein theacyl group ##STR172## wherein R represents (lower)alkyl.
 7. A compoundof claim 1 wherein the acyl group ##STR173## wherein R is (lower)alkyl.8. A compound of claim 1 wherein the acyl group ##STR174## wherein R is(lower)alkyl.
 9. The compound of claim 1 wherein the acyl group##STR175##
 10. The compound of claim 1 wherein the acyl group ##STR176##11. The compound of claim 1 wherein the acyl group ##STR177##
 12. Acompound of claim 1 wherein the acyl group ##STR178##
 13. The D isomerof the compound of claim 2.